Vaccines against HPV and HPV-related diseases

ABSTRACT

Embodiments relate to novel vaccines against human papillomavirus (HPV) and HPV-related diseases, including multiple types of cancers. The HPV vaccines are composed of anti-human dendritic cell (DC) surface receptor antibodies, including CD40, and E6/7 proteins of HPV16 and 18. The technology described is not limited to making vaccines against HPV16- and HPV18-related diseases and can be applied to making vaccines carrying E6/7 from any type of HPV. The HPV vaccines described can target DCs, major and professional antigen presenting cells (APCs), and can induce and activate potent HPV E6/7-specific and strong CD4+ and CD8+ T cell responses. The HPV vaccines can be used for the prevention of HPV infection and HPV-related diseases as well as for the treatment of HPV-related diseases, including cancers.

This application is a continuation of U.S. patent application Ser. No.15/111,357, filed Jul. 13, 2016, which is a national phase applicationunder 35 U.S.C. § 371 of International Application No.PCT/US2015/011236, filed Jan. 13, 2015, which claims the benefit ofpriority to U.S. Provisional Patent Application Ser. No. 61/926,821,filed Jan. 13, 2014, and U.S. Provisional Patent Application Ser. No.62/002,718, filed May 23, 2014, the enture contencts of each of whichare hereby incorporated by reference in their entirety.

The invention was made with government support under Grant No. U19AI057234 awarded by the National Institutes of Health and the NationalInstitute of Allergy and Infectious Diseases. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates generally to the field of medicine. Moreparticularly, it concerns new and novel vaccines against Human PapillomaVirus (HPV) and HPV-related diseases, including multiple types ofcancers.

2. Description of Related Art

Human papillomavirus (HPV) is one of the most commonsexually-transmitted pathogens. Current HPV prophylactic vaccine haveshown significant clinical efficacy in the prevention of HPV infection,but it exhibits no efficacy in the treatment of infected patients andHPV-related cancers. HPV infection causes virtually all cervicalcancers, and many anal, vaginal, vulvar, penile, and oropharyngeal(throat) cancers. Thus, the development of safe and effective vaccinesfor patients who are infected with HPV and have HPV-related cancers arein high demand. HPV infection also causes HIV-related malignancy andcancers.

Current HPV vaccines are recombinant virus-like particles made of capsid(L) proteins of HPV 6, 11, 16, and 18. These vaccines can elicit strongantibody responses and thus can prevent HPV infection. To suppress viralreplication and to eradicate HPV-related cancers, vaccines need to evokestrong T cell responses, particularly cytotoxic CD8+ lymphocytes (CTLs)that can kill virus-infected cells followed by the inhibition of HPVreplication as well as HPV-related tumor cells.

Several types of vaccine models (including peptides, proteins, andDNA-based vaccines and vaccines carried by live-attenuated vectors) havebeen tested, but these vaccines have drawbacks either in efficacy orsafety particularly in immunodeficient patients. This gap necessitatesdeveloping safe and potent immunotherapeutic vaccines againstHPV-associated cancer.

A wealth of evidence has led to the conclusion that virtually all casesof cervical cancer are attributable to persistent infection by a subsetof HPV types, especially HPV type 16 (HPV 16) and HPV type 18 (HPV 18).These HPV types also cause a proportion of other cancers of mucosa,including vulvar, vaginal, anal, penile, and oropharyngeal cancers. HPV16 is the predominant type in squamous cell carcinoma of the cervix, andHPV 18 is the second most common type with prevalence ranging from 12.6%in Central/South America to 25.7% in South Asia. In addition, HPV 18 hasbeen implicated in rapidly developing and potentially more aggressivecervical carcinomas. However, subclinical infections are the most commonmanifestation of HPV infection. Different studies reported between 15%and 36% of subclinical infections in sexually active adults.

SUMMARY OF THE INVENTION

Disclosed is a fusion protein comprising an anti-CD40 antibody orfragment thereof, comprising at least three complementarity determiningregions from an anti-CD40 antibody, at least one peptide linker, and atleast one human papillomavirus (HPV) E6 or E7 antigen, wherein the E6 orE7 antigen or antigens are HPV type 16 or HPV type 18 antigens. In someembodiments, the anti-CD40 antibody or fragment thereof comprises atleast the variable region from an anti-CD40 antibody. The variableregion can be from a light chain or heavy chain. In some embodiments,the anti-CD40 antibody or fragment thereof comprises at least thevariable region from an anti-CD40 antibody light chain and at least thevariable region from an anti-CD40 antibody heavy chain. In someembodiments, the anti-CD40 antibody or fragment thereof comprises sixCDRs from an anti-CD40 antibody. In some embodiments, the anti-CD40antibody or fragment thereof is humanized. In some embodiments, thepeptide linker or linkers are a flexible linker. In some embodiments,the peptide linker or linkers comprise one or more glycosylation sites.In some embodiments, the peptide linker or linkers are Flexv1 (SEQ IDNO:5) and/or f1 (SEQ ID NO:6). In some embodiments, the HPV antigens areE6 and E7. In some embodiments, the fusion protein comprises thesequence of SEQ ID NO:19. In some embodiments, the fusion proteincomprises the sequence of SEQ ID NO:21. In some embodiments, at leastone HPV E6 antigen is an HPV type 16 antigen and at least one HPV E6antigen is an HPV type 18 antigen. In some embodiments, at least one HPVE7 antigen is an HPV type 16 antigen and at least one HPV E7 antigen isan HPV type 18 antigen. In some embodiments, at least one HPV E6 antigenis an HPV type 16 antigen and at least one HPV E6 antigen is an HPV type18 antigen and at least one HPV E7 antigen is an HPV type 16 antigen andat least one HPV E7 antigen is an HPV type 18 antigen.

Also disclosed is a fusion protein comprising the amino acid sequencesof at least SEQ ID NOs:11-13 or SEQ ID NOs:14-16 and at least one humanpapillomavirus (HPV) E6 or E7 antigen. In some embodiments, the fusionprotein comprises SEQ ID NOs:11-13 and SEQ ID NOs:14-16. In someembodiments, the E6 or E7 antigen or antigens are HPV type 16 or HPVtype 18 antigens. In some embodiments, the fusion protein furthercomprises a peptide linker. In some embodiments, the peptide linker is aflexible linker. In some embodiments, the peptide linker comprises oneor more glycosylation sites. In some embodiments, the peptide linker isFlexv1 (SEQ ID NO:5) and/or f1 (SEQ ID NO:6).

Also disclosed is a pharmaceutical composition comprising any of theabove fusion proteins.

Also disclosed is a method of making any of the above fusion proteinscomprising isolating the fusion protein from a recombinant host cellexpressing the fusion protein.

Also disclosed is a composition comprising a dendritic cell targetingcomplex comprising an anti-CD40 antibody or fragment thereof comprisingat least three complementarity determining regions from an anti-CD40antibody, at least one peptide linker and at least one humanpapillomavirus (HPV) E6 or E7 antigen, wherein the E6 or E7 antigen orantigens are an HPV type 16 or HPV type 18 antigen. In some embodiments,the anti-CD40 antibody or fragment thereof comprises at least thevariable region from an anti-CD40 antibody. The variable region can befrom a light chain or heavy chain. In some embodiments, the anti-CD40antibody or fragment thereof comprises at least the variable region froman anti-CD40 antibody light chain and at least the variable region froman anti-CD40 antibody heavy chain. In some embodiments, the anti-CD40antibody or fragment thereof comprises six CDRs from an anti-CD40antibody. In some embodiments, the anti-CD40 antibody or fragmentthereof is humanized. In some embodiments, the peptide linker or linkersare a flexible linker. In some embodiments, the peptide linker orlinkers comprise one or more glycosylation sites. In some embodiments,the peptide linker or linkers are selected from Flexv1 (SEQ ID NO:5) orf1 (SEQ ID NO:6). In some embodiments, the HPV antigens are E6 and E7.In some embodiments, the fusion protein comprises the sequence of SEQ IDNO: 19. In some embodiments, the fusion protein comprises the sequenceof SEQ ID NO:21. In some embodiments, at least one HPV E6 antigen is anHPV type 16 antigen and at least one HPV E6 antigen is an HPV type 18antigen. In some embodiments, at least one HPV E7 antigen is an HPV type16 antigen and at least one HPV E7 antigen is an HPV type 18 antigen. Insome embodiments, at least one HPV E6 antigen is an HPV type 16 antigenand at least one HPV E6 antigen is an HPV type 18 antigen and at leastone HPV E7 antigen is an HPV type 16 antigen and at least one HPV E7antigen is an HPV type 18 antigen.

In some embodiments the HPV E6 and E7 antigens are selected from SEQ IDNO: 1-4. In other embodiments the HPV E6 and E7 antigens are at least70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100%, or any range derivabletherein, identical to any combination of SEQ ID NO: 1-4. In otherembodiments the HPV E6 and E7 antigens are at least 70%, 75%, 80%, 85%,90%, 95%, 98%, 99% or 100%, or any range derivable therein, identical toSEQ ID NO: 1-4. In still other embodiments, the HPV E6 antigen is a 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115 or 120, or anyrange derivable therein, subset of contiguous amino acids of SEQ ID NO:1 and/or 3 and the HPV E7 antigen is a 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 105, 110, 115 or 120, or any range derivable therein,subset of contiguous amino acids of SEQ ID NO: 2 and/or 4.

Also disclosed is a vector comprising a polynucleotide sequence encodinga fusion protein comprising an anti-CD40 antibody or fragment thereofcomprising at least three complementarity determining regions from ananti-CD40 antibody, at least one peptide linker and at least one humanpapillomavirus (HPV) E6 or E7 antigen, wherein the E6 or E7 antigen orantigens are HPV type 16 or HPV type 18 antigens. In some embodiments,the anti-CD40 antibody or fragment thereof comprises an anti-CD40antibody light chain variable region. In some embodiments, the anti-CD40antibody or fragment thereof comprises an anti-CD40 antibody heavy chainvariable region. In some embodiments, the polynucleotide sequenceencodes at least one HPV type 16 E6 antigen, at least one HPV type 16 E7antigen or at least one HPV type 18 E6 antigen and at least one HPV type18 E7 antigen. In some embodiments, the polynucleotide sequence encodesa polypeptide comprising SEQ ID NO: 19. In some embodiments, thepolynucleotide sequence encodes a polypeptide comprising SEQ ID NO: 21.In some embodiments, the polynucleotide sequence encodes at least oneHPV type 16 E6 antigen, at least one HPV type 16 E7 antigen, at leastone HPV type 18 E6 antigen and at least one HPV type 18 E7 antigen.

Also disclosed is a method for preventing a human papillomavirus (HPV)infection comprising administering to a patient a composition comprisinga dendritic cell targeting complex comprising an anti-CD40 antibody orfragment thereof comprising at least six complementarity determiningregions from an anti-CD40 antibody, at least one peptide linker and atleast one human papillomavirus (HPV) E6 or E7 antigen, wherein the E6 orE7 antigen or antigens are HPV type 16 or HPV type 18 antigens. In someembodiments, the anti-CD40 antibody or fragment thereof comprises ananti-CD40 antibody light chain variable region and an anti-CD40 antibodyheavy chain variable region. In some embodiments, the anti-CD40 antibodyor fragment thereof is humanized. In some embodiments, at least one HPVE6 antigen is an HPV type 16 antigen and at least one HPV E7 antigen isan HPV type 16 antigen. In some embodiments, at least one HPV E6 antigenis an HPV type 18 antigen and at least one HPV E7 antigen is an HPV type18 antigen. In some embodiments, at least one HPV E6 antigen is an HPVtype 16 antigen, at least one HPV E7 antigen is an HPV type 16 antigen,at least one HPV E6 antigen is an HPV type 18 antigen and at least oneHPV E7 antigen is an HPV type 18 antigen. In some embodiments, thedendritic cell targeting complex comprises SEQ ID NO: 19. In someembodiments, dendritic cell targeting complex comprises SEQ ID NO: 21.In some embodiments, the composition further comprises an adjuvant. Insome embodiments, the method further comprises administering to thepatient a separate HPV vaccine. In some embodiments, the separate HPVvaccine is Gardasil™ or Cervarix™.

Also disclosed is a method for treating a human papillomavirus (HPV)infection comprising administering to a patient a composition comprisinga dendritic cell targeting complex comprising an anti-CD40 antibody orfragment thereof comprising at least six complementarity determiningregions from an anti-CD40 antibody, at least one peptide linker and atleast one human papillomavirus (HPV) E6 or E7 antigen, wherein the E6 orE7 antigen or antigens are HPV type 16 or HPV type 18 antigens. In someembodiments, the anti-CD40 antibody or fragment thereof comprises ananti-CD40 antibody light chain variable region and an anti-CD40 antibodyheavy chain variable region. In some embodiments, the anti-CD40 antibodyor fragment thereof is humanized. In some embodiments, at least one HPVE6 antigen is an HPV type 16 antigen and at least one HPV E7 antigen isan HPV type 16 antigen. In some embodiments, at least one HPV E6 antigenis an HPV type 18 antigen and at least one HPV E7 antigen is an HPV type18 antigen. In some embodiments, at least one HPV E6 antigen is an HPVtype 16 antigen, at least one HPV E7 antigen is an HPV type 16 antigen,at least one HPV E6 antigen is an HPV type 18 antigen and at least oneHPV E7 antigen is an HPV type 18 antigen. In some embodiments, thedendritic cell targeting complex comprises SEQ ID NO: 19. In someembodiments, the dendritic cell targeting complex comprises SEQ ID NO:21. In some embodiments, the method further comprises administering tothe patient a separate HPV treatment.

Also disclosed is a method for inducing an immune response to at leastone HPV epitope comprising administering to a patient a compositioncomprising a dendritic cell targeting complex comprising an anti-CD40antibody or fragment thereof comprising at least six complementaritydetermining regions from an anti-CD40 antibody, at least one peptidelinker and at least one human papillomavirus (HPV) E6 or E7 antigen,wherein the E6 or E7 antigen or antigens are HPV type 16 or HPV type 18antigens. In some embodiments, the anti-CD40 antibody or fragmentthereof comprises an anti-CD40 antibody light chain variable region andan anti-CD40 antibody heavy chain variable region. In some embodiments,the anti-CD40 antibody or fragment thereof is humanized. In someembodiments, at least one HPV E6 antigen is an HPV type 16 antigen andat least one HPV E7 antigen is an HPV type 16 antigen. In someembodiments, at least one HPV E6 antigen is an HPV type 18 antigen andat least one HPV E7 antigen is an HPV type 18 antigen. In someembodiments, at least one HPV E6 antigen is an HPV type 16 antigen, atleast one HPV E7 antigen is an HPV type 16 antigen, at least one HPV E6antigen is an HPV type 18 antigen and at least one HPV E7 antigen is anHPV type 18 antigen. In some embodiments, the dendritic cell targetingcomplex comprises SEQ ID NO: 19. In some embodiments, the dendritic celltargeting complex comprises SEQ ID NO: 21. In some embodiments, thecomposition further comprises an adjuvant. In some embodiments, themethod further comprises administering to the patient a separate HPVvaccine. In some embodiments, the separate HPV vaccine is Gardasil™ orCervarix™.

Also disclosed is a method for potentiating an immune response to atleast one HPV epitope comprising administering to a patient acomposition comprising a dendritic cell targeting complex comprising ananti-CD40 antibody or fragment thereof comprising at least sixcomplementarity determining regions from an anti-CD40 antibody, at leastone peptide linker and at least one human papillomavirus (HPV) E6 or E7antigen, wherein the E6 or E7 antigen or antigens are HPV type 16 or HPVtype 18 antigens. In some embodiments, the anti-CD40 antibody orfragment thereof comprises an anti-CD40 antibody light chain variableregion and an anti-CD40 antibody heavy chain variable region. In someembodiments, the anti-CD40 antibody or fragment thereof is humanized. Insome embodiments, at least one HPV E6 antigen is an HPV type 16 antigenand at least one HPV E7 antigen is an HPV type 16 antigen. In someembodiments, at least one HPV E6 antigen is an HPV type 18 antigen andat least one HPV E7 antigen is an HPV type 18 antigen. In someembodiments, at least one HPV E6 antigen is an HPV type 16 antigen, atleast one HPV E7 antigen is an HPV type 16 antigen, at least one HPV E6antigen is an HPV type 18 antigen and at least one HPV E7 antigen is anHPV type 18 antigen. In some embodiments, the dendritic cell targetingcomplex comprises SEQ ID NO: 19. In some embodiments, the dendritic celltargeting complex comprises SEQ ID NO: 21. In some embodiments,potentiating an immune response is directed towards potentiating orincreasing or enhancing memory T-cells. In some embodiments, the methodfurther comprises administering to the patient a separate HPV treatment.

Also disclosed is a method for preventing a human papillomavirus (HPV)related disease comprising administering to a patient a compositioncomprising a dendritic cell targeting complex comprising an anti-CD40antibody or fragment thereof comprising at least six complementaritydetermining regions from an anti-CD40 antibody, at least one peptidelinker and at least one human papillomavirus (HPV) E6 or E7 antigen,wherein the E6 or E7 antigen or antigens are HPV type 16 or HPV type 18antigens. In some embodiments, the anti-CD40 antibody or fragmentthereof comprises an anti-CD40 antibody light chain variable region andan anti-CD40 antibody heavy chain variable region. In some embodiments,the anti-CD40 antibody or fragment thereof is humanized. In someembodiments, at least one HPV E6 antigen is an HPV type 16 antigen andat least one HPV E7 antigen is an HPV type 16 antigen. In someembodiments, at least one HPV E6 antigen is an HPV type 18 antigen andat least one HPV E7 antigen is an HPV type 18 antigen. In someembodiments, at least one HPV E6 antigen is an HPV type 16 antigen, atleast one HPV E7 antigen is an HPV type 16 antigen, at least one HPV E6antigen is an HPV type 18 antigen and at least one HPV E7 antigen is anHPV type 18 antigen. In some embodiments, the dendritic cell targetingcomplex comprises SEQ ID NO: 19. In some embodiments, the dendritic celltargeting complex comprises SEQ ID NO: 21. In some embodiments, thecomposition further comprises an adjuvant. In some embodiments, the HPVrelated disease is dysplasia, benign neoplasia, pre-malignant neoplasiaor cancer. In some embodiments, the HPV related disease is cancer. Insome embodiments, the cancer is cancer of the cervix, vulva, vagina,penis, anus, oropharynx, throat or lung. In some embodiments, the methodfurther comprises administering to the patient a separate HPV vaccine.In some embodiments, the separate HPV vaccine is Gardasil™ or Cervarix™.

Also disclosed is a method for treating a human papillomavirus (HPV)related disease comprising administering to a patient a compositioncomprising a dendritic cell targeting complex comprising an anti-CD40antibody or fragment thereof comprising at least six complementaritydetermining regions from an anti-CD40 antibody, at least one peptidelinker and at least one human papillomavirus (HPV) E6 or E7 antigen,wherein the E6 or E7 antigen or antigens are HPV type 16 or HPV type 18antigens. In some embodiments, the anti-CD40 antibody or fragmentthereof comprises an anti-CD40 antibody light chain variable region andan anti-CD40 antibody heavy chain variable region. In some embodiments,the anti-CD40 antibody or fragment thereof is humanized. In someembodiments, at least one HPV E6 antigen is an HPV type 16 antigen andat least one HPV E7 antigen is an HPV type 16 antigen. In someembodiments, at least one HPV E6 antigen is an HPV type 18 antigen andat least one HPV E7 antigen is an HPV type 18 antigen. In someembodiments, at least one HPV E6 antigen is an HPV type 16 antigen, atleast one HPV E7 antigen is an HPV type 16 antigen, at least one HPV E6antigen is an HPV type 18 antigen and at least one HPV E7 antigen is anHPV type 18 antigen. In some embodiments, the dendritic cell targetingcomplex comprises SEQ ID NO: 19. In some embodiments, the dendritic celltargeting complex comprises SEQ ID NO: 21. In some embodiments, the HPVrelated disease is dysplasia, benign neoplasia, pre-malignant neoplasiaor cancer. In some embodiments, the HPV related disease is cancer. Insome embodiments, the cancer is cancer of the cervix, vulva, vagina,penis, anus, oropharynx, throat or lung. In yet further embodiments, thecancer is head and neck cancer. In some embodiments, the method furthercomprises administering to the patient a separate treatment. In someembodiments, the method further comprises administering to the patient acancer treatment.

Also disclosed is a method of inhibiting HPV-infected cells in a patientcomprising administering to the patient an effective amount of acomposition comprising any of the above fusion proteins or vectors. Insome embodiments, the HPV-infected cells are in a tumor.

Also disclosed is a method of reducing the size or mass of a tumor in apatient that is suffering from an HPV infection or the tumor comprisesHPV-infected cells, comprising administering to the patient an effectiveamount of a composition comprising any of the above fusion proteins orvectors. The percent reduction in size or mass of the tumor or thepercent regression of the tumor during or following treatment may be atleast 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% 75%,80%, 85%, 90%, 95% to 100% or any derivable range therein. The percentreduction in size or mass of the tumor or the percent regression of thetumor may be such that the tumor size eases discomfort and improves thepatient's quality of life or leads to, or is associated with, aclinically favorable outcome.

Disclosed is a method of extending survival or a patient or subjectsuffering from an HPV related disease comprising administering to apatient a composition comprising a dendritic cell targeting complexcomprising an anti-CD40 antibody or fragment thereof comprising at leastsix complementarity determining regions from an anti-CD40 antibody, atleast one peptide linker and at least one human papillomavirus (HPV) E6or E7 antigen, wherein the E6 or E7 antigen or antigens are HPV type 16or HPV type 18 antigens. In some embodiments, the anti-CD40 antibody orfragment thereof comprises an anti-CD40 antibody light chain variableregion and an anti-CD40 antibody heavy chain variable region. In someembodiments, the anti-CD40 antibody or fragment thereof is humanized. Insome embodiments, at least one HPV E6 antigen is an HPV type 16 antigenand at least one HPV E7 antigen is an HPV type 16 antigen. In someembodiments, at least one HPV E6 antigen is an HPV type 18 antigen andat least one HPV E7 antigen is an HPV type 18 antigen. In someembodiments, at least one HPV E6 antigen is an HPV type 16 antigen, atleast one HPV E7 antigen is an HPV type 16 antigen, at least one HPV E6antigen is an HPV type 18 antigen and at least one HPV E7 antigen is anHPV type 18 antigen. In some embodiments, the dendritic cell targetingcomplex comprises SEQ ID NO: 19. In some embodiments, the dendritic celltargeting complex comprises SEQ ID NO: 21. In some embodiments, the HPVrelated disease is dysplasia, benign neoplasia, pre-malignant neoplasiaor cancer. In some embodiments, the HPV related disease is cancer. Insome embodiments, the cancer is cancer of the cervix, vulva, vagina,penis, anus, oropharynx, throat or lung. In yet further embodiments, thecancer is head and neck cancer. In some embodiments, the method furthercomprises administering to the patient a separate treatment. In someembodiments, the method further comprises administering to the patient acancer treatment. In some aspects, survival is extended by a period of1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days, or any range derivable therein. Insome aspects, survival is extended by a period of 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 weeks, or any range derivable therein. In some aspects,survival is extended by a period of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10months, or any range derivable therein. In some aspects, survival isextended by a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 20, 25 or 30 years, or any range derivable therein.

Any of the methods disclosed above may be implemented using any of thefusion proteins, compositions, and/or vectors disclosed above.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1: Scheme for development of recombinant anti-DC receptor-E6/E7vaccines.

FIG. 2: SDS-PAGE analysis of protein A affinity purified anti-DCreceptor antibody fused to HPV16 E6 and E7 antigens.

FIG. 3: Anti-CD40-HPV16.E6/7 can efficiently bind to human blood DCs.

FIG. 4: Anti-CD40-HPV16.E6/7 can elicit HPV16.E6/7-specific CD4+ andCD8+ T-cell responses.

FIG. 5: Anti-CD40-HPV16.E6/7 can efficiently induce HPV16.E6/7-specificCD4+ and CD8+ T-cell responses.

FIG. 6: Anti-CD40-HPV16.E6/7 can suppress TC-1 tumor progression inhuman CD40Tg mice.

FIG. 7: Effect of adjuvant co-administration with anti-CD40-HPV16.E6/7.

FIG. 8: CD40HVac plus poly IC induces E6/7-specific CD8+ T cells inhCD40 transgenic animals.

FIG. 9: CD40HVac plus poly IC induces therapeutic immunity in hCD40Tgmice. (a) Survival curves. 10 mice per group. (b) TC-1 tumorprogression. 10 mice per group.

FIG. 10: The percentage of E7-specific tetramer+CD8+ T cells in tumors,but not blood, inversely correlates with tumor volume.

FIG. 11: CD40HVac made with anti-CD40 (clone 12E12) is more efficientthan that made with anti-CD40 (clone 12B6) at inducing E6/7-specificCD8+ T cell responses.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

As described in the Examples, an anti-CD40 antibody to which an HPV E6and E7 antigen has been fused (called hereafter ‘anti-CD40-HPV 16.E6/7’)has been shown to induce a strong immune response against said antigens(including a strong T-cell response). This provides an efficient andeffective method of eliciting and potentiating an immune response to HPVantigens. Moreover, an anti-CD40-HPV16.E6/7 has been used in aprime-boost strategy in combination with a poly IC adjuvant to suppressTC-1 tumor progression in human CD40 transgenic mice. Thus, it has beendemonstrated that said anti-CD40-HPV16.E6/7 can elicit E6/E7-specificCD8+ cytotoxic T lymphocytes and when administered with a poly ICadjuvant, serves as an efficient vaccine.

I. NUCLEIC ACIDS

In certain embodiments, there are recombinant nucleic acids encoding theproteins, polypeptides, or peptides described herein. Polynucleotidescontemplated for use in methods and compositions include those encodingantibodies to DC receptors or binding portions thereof, HPV antigens,linker regions or adjuvants.

As used in this application, the term “polynucleotide” refers to anucleic acid molecule that either is recombinant or has been isolatedfree of total genomic nucleic acid. Included within the term“polynucleotide” are oligonucleotides (nucleic acids 100 residues orfewer in length), recombinant vectors, including, for example, plasmids,cosmids, phage, viruses, and the like. Polynucleotides include, incertain aspects, regulatory sequences, isolated substantially away fromtheir naturally occurring genes or protein encoding sequences.Polynucleotides may be single-stranded (coding or antisense) ordouble-stranded, and may be RNA, DNA (genomic, cDNA or synthetic),analogs thereof, or a combination thereof. Additional coding ornon-coding sequences may, but need not, be present within apolynucleotide.

In this respect, the term “gene,” “polynucleotide,” or “nucleic acid” isused to refer to a nucleic acid that encodes a protein, polypeptide, orpeptide (including any sequences required for proper transcription,post-translational modification, or localization). As will be understoodby those in the art, this term encompasses genomic sequences, expressioncassettes, cDNA sequences, and smaller engineered nucleic acid segmentsthat express, or may be adapted to express, proteins, polypeptides,domains, peptides, fusion proteins, and mutants. A nucleic acid encodingall or part of a polypeptide may contain a contiguous nucleic acidsequence encoding all or a portion of such a polypeptide. It also iscontemplated that a particular polypeptide may be encoded by nucleicacids containing variations having slightly different nucleic acidsequences but, nonetheless, encode the same or substantially similarprotein (see above).

In particular embodiments, there are isolated nucleic acid segments andrecombinant vectors incorporating nucleic acid sequences that encodepolypeptides (e.g., an antibody or fragment thereof) that bind to DCreceptors, are HPV antigens, are linker regions or are fusion proteinscomprising any combination of a DC receptor antibody or antibodies orfragments thereof, HPV antigens (such as E6 or E7 from any HPV type) andlinker regions. The term “recombinant” may be used in conjunction with apolypeptide or the name of a specific polypeptide, and this generallyrefers to a polypeptide produced from a nucleic acid molecule that hasbeen manipulated in vitro or that is a replication product of such amolecule.

The nucleic acid segments, regardless of the length of the codingsequence itself, may be combined with other nucleic acid sequences, suchas promoters, polyadenylation signals, additional restriction enzymesites, multiple cloning sites, other coding segments, and the like, suchthat their overall length may vary considerably. It is thereforecontemplated that a nucleic acid fragment of almost any length may beemployed, with the total length preferably being limited by the ease ofpreparation and use in the intended recombinant nucleic acid protocol.In some cases, a nucleic acid sequence may encode a polypeptide sequencewith additional heterologous coding sequences, for example to allow forpurification of the polypeptide, transport, secretion,post-translational modification, or for therapeutic benefits such astargeting or efficacy. As discussed above, a tag or other heterologouspolypeptide may be added to the modified polypeptide-encoding sequence,wherein “heterologous” refers to a polypeptide that is not the same asthe modified polypeptide.

In certain embodiments, there are polynucleotide variants havingsubstantial identity to the sequences disclosed herein; those comprisingat least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or highersequence identity, including all values and ranges there between,compared to a polynucleotide sequence provided herein using the methodsdescribed herein (e.g., BLAST analysis using standard parameters). Incertain aspects, the isolated polynucleotide will comprise a nucleotidesequence encoding a polypeptide that has at least 90%, preferably 95%and above, identity to an amino acid sequence described herein, over theentire length of the sequence; or a nucleotide sequence complementary tosaid isolated polynucleotide.

Vectors

Polypeptides may be encoded by a nucleic acid molecule. The nucleic acidmolecule can be in the form of a nucleic acid vector. The term “vector”is used to refer to a carrier nucleic acid molecule into which aheterologous nucleic acid sequence can be inserted for introduction intoa cell where it can be replicated and expressed. A nucleic acid sequencecan be “heterologous,” which means that it is in a context foreign tothe cell in which the vector is being introduced or to the nucleic acidin which is incorporated, which includes a sequence homologous to asequence in the cell or nucleic acid but in a position within the hostcell or nucleic acid where it is ordinarily not found. Vectors includeDNAs, RNAs, plasmids, cosmids, viruses (bacteriophage, animal viruses,and plant viruses), and artificial chromosomes (e.g., YACs). One ofskill in the art would be well equipped to construct a vector throughstandard recombinant techniques (for example Sambrook et al., 2001;Ausubel et al., 1996, both incorporated herein by reference). Vectorsmay be used in a host cell to produce an antibody or fragment thereofthat binds a dendritic cell receptor, an HPV antigen or antigens (e.g.E6 and/or E7 from one or multiple HPV types), a linker or multiplelinker regions, an adjuvant or multiple adjuvants, any combination ofthe aforementioned proteins or a fusion protein or fusion proteinscomprising any combination of the aforementioned proteins.

The term “expression vector” refers to a vector containing a nucleicacid sequence coding for at least part of a gene product capable ofbeing transcribed. In some cases, RNA molecules are then translated intoa protein, polypeptide, or peptide. Expression vectors can contain avariety of “control sequences,” which refer to nucleic acid sequencesnecessary for the transcription and possibly translation of an operablylinked coding sequence in a particular host organism. In addition tocontrol sequences that govern transcription and translation, vectors andexpression vectors may contain nucleic acid sequences that serve otherfunctions as well and are described herein.

Host Cells

As used herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. All of these terms also include their progeny,which is any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.In the context of expressing a heterologous nucleic acid sequence, “hostcell” refers to a prokaryotic or eukaryotic cell, and it includes anytransformable organism that is capable of replicating a vector orexpressing a heterologous gene encoded by a vector. A host cell can, andhas been, used as a recipient for vectors or viruses. A host cell may be“transfected” or “transformed,” which refers to a process by whichexogenous nucleic acid, such as a recombinant protein-encoding sequence,is transferred or introduced into the host cell. A transformed cellincludes the primary subject cell and its progeny.

Some vectors may employ control sequences that allow it to be replicatedand/or expressed in both prokaryotic and eukaryotic cells. One of skillin the art would further understand the conditions under which toincubate all of the above described host cells to maintain them and topermit replication of a vector. Also understood and known are techniquesand conditions that would allow large-scale production of vectors, aswell as production of the nucleic acids encoded by vectors and theircognate polypeptides, proteins, or peptides.

Expression Systems

Numerous expression systems exist that comprise at least a part or allof the compositions discussed above. Prokaryote- and/or eukaryote-basedsystems can be employed for use with an embodiment to produce nucleicacid sequences, or their cognate polypeptides, proteins and peptides.Many such systems are commercially and widely available.

The insect cell/baculovirus system can produce a high level of proteinexpression of a heterologous nucleic acid segment, such as described inU.S. Pat. Nos. 5,871,986, 4,879,236, both herein incorporated byreference, and which can be bought, for example, under the name MAXBAC®2.0 from INVITROGEN® and BACPACK™ BACULOVIRUS EXPRESSION SYSTEM FROMCLONTECH®.

In addition to the disclosed expression systems, other examples ofexpression systems include STRATAGENE®'s COMPLETE CONTROL InducibleMammalian Expression System, which involves a syntheticecdysone-inducible receptor, or its pET Expression System, an E. coliexpression system. Another example of an inducible expression system isavailable from INVITROGEN®, which carries the T-REX™(tetracycline-regulated expression) System, an inducible mammalianexpression system that uses the full-length CMV promoter. INVITROGEN®also provides a yeast expression system called the Pichia methanolicaExpression System, which is designed for high-level production ofrecombinant proteins in the methylotrophic yeast Pichia methanolica. Oneof skill in the art would know how to express a vector, such as anexpression construct, to produce a nucleic acid sequence or its cognatepolypeptide, protein, or peptide.

II. PROTEINACEOUS COMPOSITIONS

Substitutional variants typically contain the exchange of one amino acidfor another at one or more sites within the protein, and may be designedto modulate one or more properties of the polypeptide, with or withoutthe loss of other functions or properties. Substitutions may beconservative, that is, one amino acid is replaced with one of similarshape and charge. Conservative substitutions are well known in the artand include, for example, the changes of: alanine to serine; arginine tolysine; asparagine to glutamine or histidine; aspartate to glutamate;cysteine to serine; glutamine to asparagine; glutamate to aspartate;glycine to proline; histidine to asparagine or glutamine; isoleucine toleucine or valine; leucine to valine or isoleucine; lysine to arginine;methionine to leucine or isoleucine; phenylalanine to tyrosine, leucineor methionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine. Alternatively, substitutions may benon-conservative such that a function or activity of the polypeptide isaffected. Non-conservative changes typically involve substituting aresidue with one that is chemically dissimilar, such as a polar orcharged amino acid for a nonpolar or uncharged amino acid, and viceversa.

Proteins may be recombinant, or synthesized in vitro. Alternatively, anon-recombinant or recombinant protein may be isolated from bacteria. Itis also contemplated that a bacteria containing such a variant may beimplemented in compositions and methods. Consequently, a protein neednot be isolated.

The term “functionally equivalent codon” is used herein to refer tocodons that encode the same amino acid, such as the six codons forarginine or serine, and also refers to codons that encode biologicallyequivalent amino acids (see Table, below).

Codon Table Amino Acids Codons Alanine Ala A GCA GCC GCG GCU CysteineCys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

It also will be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids, or 5′ or 3′ sequences, respectively, and yet still beessentially as set forth in one of the sequences disclosed herein, solong as the sequence meets the criteria set forth above, including themaintenance of biological protein activity where protein expression isconcerned. The addition of terminal sequences particularly applies tonucleic acid sequences that may, for example, include various non-codingsequences flanking either of the 5′ or 3′ portions of the coding region.

The following is a discussion based upon changing of the amino acids ofa protein to create an equivalent, or even an improved,second-generation molecule. For example, certain amino acids may besubstituted for other amino acids in a protein structure withoutappreciable loss of interactive binding capacity with structures suchas, for example, antigen-binding regions of antibodies or binding siteson substrate molecules or loss of antigenicity in antigenic peptides orproteins. Since it is the interactive capacity and nature of a proteinthat defines that protein's biological functional activity, certainamino acid substitutions can be made in a protein sequence, and in itsunderlying DNA coding sequence, and nevertheless produce a protein withlike properties. It is thus contemplated by the inventors that variouschanges may be made in the DNA sequences of genes without appreciableloss of their biological utility or activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982). It is accepted thatthe relative hydropathic character of the amino acid contributes to thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like.

It also is understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein. It is understood that an amino acid can besubstituted for another having a similar hydrophilicity value and stillproduce a biologically equivalent and immunologically equivalentprotein.

As outlined above, amino acid substitutions generally are based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take into consideration the variousforegoing characteristics are well known and include: arginine andlysine; glutamate and aspartate; serine and threonine; glutamine andasparagine; and valine, leucine and isoleucine.

It is contemplated that in compositions there is between about 0.001 mgand about 10 mg of total polypeptide, peptide, and/or protein per ml.Thus, the concentration of protein (including a fusion protein) in acomposition can be about, at least about or at most about 0.001, 0.010,0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5,3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5,10.0 mg/ml or more (or any range derivable therein). Of this, about, atleast about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% may be anantibody that binds DC receptor or a fusion protein comprising anantibody that binds a DC receptor, and may be used in combination withother HPV antigens, linker regions or adjuvants described herein.

Polypeptides and Polypeptide Production

Embodiments involve polypeptides, peptides, and proteins and immunogenicfragments thereof for use in various aspects described herein. Forexample, specific antibodies are assayed for or used in binding to DCreceptors and presenting HPV antigens. In specific embodiments, all orpart of proteins described herein can also be synthesized in solution oron a solid support in accordance with conventional techniques. Variousautomatic synthesizers are commercially available and can be used inaccordance with known protocols. See, for example, Stewart and Young,(1984); Tam et al., (1983); Merrifield, (1986); and Barany andMerrifield (1979), each incorporated herein by reference. Alternatively,recombinant DNA technology may be employed wherein a nucleotide sequencethat encodes a peptide or polypeptide is inserted into an expressionvector, transformed or transfected into an appropriate host cell andcultivated under conditions suitable for expression.

One embodiment includes the use of gene transfer to cells, includingmicroorganisms, for the production and/or presentation of proteins. Thegene for the protein of interest may be transferred into appropriatehost cells followed by culture of cells under the appropriateconditions. A nucleic acid encoding virtually any polypeptide may beemployed. The generation of recombinant expression vectors, and theelements included therein, are discussed herein. Alternatively, theprotein to be produced may be an endogenous protein normally synthesizedby the cell used for protein production.

In a certain aspects an DC receptor or receptor fragment comprisessubstantially all of the extracellular domain of a protein which has atleast 85% identity, at least 90% identity, at least 95% identity, or atleast 97-99% identity, including all values and ranges there between, toa sequence selected over the length of the fragment sequence.

Also included in immunogenic compositions are fusion proteins composedof HPV antigens, or immunogenic fragments of HPV antigens (e.g., E6 orE7). HPV antigens may be from any type (e.g. type 16 or type 18). Forexample an HPV antigen may be selected from a single or combination ofHPV type. An HPV antigen or combination of HPV antigen may be from HPVtype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120.Alternatively, embodiments also include individual fusion proteins ofHPV proteins or immunogenic fragments thereof or anti-DC receptorantibodies or fragments thereof, as a fusion protein with heterologoussequences such as a provider of T-cell epitopes or purification tags,for example: β-galactosidase, glutathione-S-transferase, greenfluorescent proteins (GFP), epitope tags such as FLAG, myc tag, polyhistidine, or viral surface proteins such as influenza virushaemagglutinin, or bacterial proteins such as tetanus toxoid, diphtheriatoxoid, CRM197.

Antibodies and Antibody-Like Molecules

In certain aspects, one or more antibodies or antibody-like molecules(e.g., polypeptides comprising antibody CDR domains) may be obtained orproduced which have a specificity for a DC receptor. These antibodiesmay be used in various diagnostic or therapeutic applications describedherein.

As used herein, the term “antibody” is intended to refer broadly to anyimmunologic binding agent such as IgG, IgM, IgA, IgD and IgE as well aspolypeptides comprising antibody CDR domains that retain antigen bindingactivity. Thus, the term “antibody” is used to refer to anyantibody-like molecule that has an antigen binding region, and includesantibody fragments such as Fab′, Fab, F(ab′)2, single domain antibodies(DABs), Fv, scFv (single chain Fv), and polypeptides with antibody CDRs,scaffolding domains that display the CDRs (e.g., anticalins) or ananobody. For example, the nanobody can be antigen-specific VHH (e.g., arecombinant VHH) from a camelid IgG2 or IgG3, or a CDR-displaying framefrom such camelid Ig. The techniques for preparing and using variousantibody-based constructs and fragments are well known in the art. Meansfor preparing and characterizing antibodies are also well known in theart (See, e.g., Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988; incorporated herein by reference).

“Mini-antibodies” or “minibodies” are also contemplated for use withembodiments. Minibodies are sFv polypeptide chains which includeoligomerization domains at their C-termini, separated from the sFv by ahinge region (Pack, et al., 1992). The oligomerization domain comprisesself-associating a-helices, e.g., leucine zippers, that can be furtherstabilized by additional disulfide bonds. The oligomerization domain isdesigned to be compatible with vectorial folding across a membrane, aprocess thought to facilitate in vivo folding of the polypeptide into afunctional binding protein. Generally, minibodies are produced usingrecombinant methods well known in the art. See, e.g., Pack et al.(1992); Cumber et al. (1992).

Antibody-like binding peptidomimetics are also contemplated inembodiments. Liu et al., 2003 describe “antibody like bindingpeptidomimetics” (ABiPs), which are peptides that act as pared-downantibodies and have certain advantages of longer serum half-life as wellas less cumbersome synthesis methods.

Alternative scaffolds for antigen binding peptides, such as CDRs arealso available and can be used to generate DC receptor-binding moleculesin accordance with the embodiments. Generally, a person skilled in theart knows how to determine the type of protein scaffold on which tograft at least one of the CDRs arising from the original antibody. Moreparticularly, it is known that to be selected such scaffolds must meetthe greatest number of criteria as follows (Skerra, 2000): goodphylogenetic conservation; known three-dimensional structure (as, forexample, by crystallography, NMR spectroscopy or any other techniqueknown to a person skilled in the art); small size; few or nopost-transcriptional modifications; and/or easy to produce, express andpurify.

The origin of such protein scaffolds can be, but is not limited to, thestructures selected among: fibronectin and preferentially fibronectintype III domain 10, lipocalin, anticalin (Skerra, 2001), protein Zarising from domain B of protein A of Staphylococcus aureus, thioredoxinA or proteins with a repeated motif such as the “ankyrin repeat” (Kohlet al., 2003), the “armadillo repeat”, the “leucine-rich repeat” and the“tetratricopeptide repeat”. For example, anticalins or lipocalinderivatives are a type of binding proteins that have affinities andspecificities for various target molecules and can be used as DCreceptor-binding molecules. Such proteins are described in US PatentPublication Nos. 20100285564, 20060058510, 20060088908, 20050106660, andPCT Publication No. WO2006/056464, incorporated herein by reference.

Scaffolds derived from toxins such as, for example, toxins fromscorpions, insects, plants, mollusks, etc., and the protein inhibitersof neuronal NO synthase (PIN) may also be used in certain aspects.

Monoclonal antibodies (MAbs) are recognized to have certain advantages,e.g., reproducibility and large-scale production. Embodiments includemonoclonal antibodies of the human, murine, monkey, rat, hamster, rabbitand chicken origin.

“Humanized” antibodies are also contemplated, as are chimeric antibodiesfrom mouse, rat, or other species, bearing human constant and/orvariable region domains, bispecific antibodies, recombinant andengineered antibodies and fragments thereof. As used herein, the term“humanized” immunoglobulin refers to an immunoglobulin comprising ahuman framework region and one or more CDR's from a non-human (usually amouse or rat) immunoglobulin. The non-human immunoglobulin providing theCDR's is called the “donor” and the human immunoglobulin providing theframework is called the “acceptor”. A “humanized antibody” is anantibody comprising a humanized light chain and a humanized heavy chainimmunoglobulin. In order to describe antibodies of some embodiments, thestrength with which an antibody molecule binds an epitope, known asaffinity, can be measured. The affinity of an antibody may be determinedby measuring an association constant (Ka) or dissociation constant(Kd).Antibodies deemed useful in certain embodiments may have an associationconstant of about, at least about, or at most about 10e6, 10e7,10e8,10e9 or 10e10 M or any range derivable therein. Similarly, in someembodiments antibodies may have a dissociation constant of about, atleast about or at most about 10e-6, 10e-7, 10e-8, 10e-9 or 10e-10. M orany range derivable therein. These values are reported for antibodiesdiscussed herein and the same assay may be used to evaluate the bindingproperties of such antibodies

In certain embodiments, a polypeptide that specifically binds to DCreceptors is able to bind a DC receptor on the surface of the cells andpresent an HPV antigen that allows the generation of a robust immuneresponse. Moreover, in some embodiments, the polypeptide that is usedcan provided protective immunity against HPV or provides a means oftherapy for HPV by generating a robust immune response.

1. Methods for Generating Antibodies

Methods for generating antibodies (e.g., monoclonal antibodies and/ormonoclonal antibodies) are known in the art. Briefly, a polyclonalantibody is prepared by immunizing an animal with a DC receptorpolypeptide or a portion thereof in accordance with embodiments andcollecting antisera from that immunized animal.

A wide range of animal species can be used for the production ofantisera. Typically the animal used for production of antisera is arabbit, a mouse, a rat, a hamster, a guinea pig or a goat. The choice ofanimal may be decided upon the ease of manipulation, costs or thedesired amount of sera, as would be known to one of skill in the art. Itwill be appreciated that antibodies can also be produced transgenicallythrough the generation of a mammal or plant that is transgenic for theimmunoglobulin heavy and light chain sequences of interest andproduction of the antibody in a recoverable form therefrom. Inconnection with the transgenic production in mammals, antibodies can beproduced in, and recovered from, the milk of goats, cows, or othermammals. See, e.g., U.S. Pat. Nos. 5,827,690, 5,756,687, 5,750,172, and5,741,957.

As is also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Suitableadjuvants include any acceptable immunostimulatory compound, such ascytokines, chemokines, cofactors, toxins, plasmodia, syntheticcompositions or vectors encoding such adjuvants.

Adjuvants that may be used in accordance with embodiments include, butare not limited to, IL-1, IL-2, IL-4, IL-7, IL-12, -interferon, GMCSP,BCG, aluminum hydroxide, MDP compounds, such as thur-MDP and nor-MDP,CGP (MTP-PE), lipid A, poly IC, montaninde, GMCSF, and monophosphoryllipid A (MPL). RIBI, which contains three components extracted frombacteria, MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS)in a 2% squalene/Tween 80 emulsion is also contemplated. MHC antigensmay even be used. Exemplary adjuvants may include complete Freund'sadjuvant (a non-specific stimulator of the immune response containingkilled Mycobacterium tuberculosis), incomplete Freund's adjuvants and/oraluminum hydroxide adjuvant.

In addition to adjuvants, it may be desirable to coadminister biologicresponse modifiers (BRM), which have been shown to upregulate T cellimmunity or downregulate suppressor cell activity. Such BRMs include,but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, Pa.);low-dose Cyclophosphamide (CYP; 300 mg/m2) (Johnson/Mead, N.J.),cytokines such as -interferon, IL-2, or IL-12 or genes encoding proteinsinvolved in immune helper functions, such as B-7.

The amount of immunogen composition used in the production of antibodiesvaries upon the nature of the immunogen as well as the animal used forimmunization. A variety of routes can be used to administer theimmunogen including but not limited to subcutaneous, intramuscular,intradermal, intraepidermal, intravenous, intratumoral andintraperitoneal. The production of antibodies may be monitored bysampling blood of the immunized animal at various points followingimmunization.

MAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified protein, polypeptide, peptide or domain, be it awild-type or mutant composition. The immunizing composition isadministered in a manner effective to stimulate antibody producingcells.

The methods for generating monoclonal antibodies (MAbs) generally beginalong the same lines as those for preparing polyclonal antibodies. Insome embodiments, Rodents such as mice and rats are used in generatingmonoclonal antibodies. In some embodiments, rabbit, sheep or frog cellsare used in generating monoclonal antibodies. The use of rats is wellknown and may provide certain advantages (Goding, 1986, pp. 60 61). Mice(e.g., BALB/c mice) are routinely used and generally give a highpercentage of stable fusions.

MAbs produced by either means may be further purified, if desired, usingfiltration, centrifugation and various chromatographic methods such asHPLC or affinity chromatography. Fragments of the monoclonal antibodiescan be obtained from the monoclonal antibodies so produced by methodswhich include digestion with enzymes, such as pepsin or papain, and/orby cleavage of disulfide bonds by chemical reduction. Alternatively,monoclonal antibody fragments can be synthesized using an automatedpeptide synthesizer.

It is also contemplated that a molecular cloning approach may be used togenerate monoclonal antibodies. In one embodiment, combinatorialimmunoglobulin phagemid libraries are prepared from RNA isolated fromthe spleen of the immunized animal, and phagemids expressing appropriateantibodies are selected by panning using cells expressing the antigenand control cells. The advantages of this approach over conventionalhybridoma techniques are that approximately 104 times as many antibodiescan be produced and screened in a single round, and that newspecificities are generated by H and L chain combination which furtherincreases the chance of finding appropriate antibodies.

Another embodiment concerns producing antibodies, for example, as isfound in U.S. Pat. No. 6,091,001, which describes methods to produce acell expressing an antibody from a genomic sequence of the cellcomprising a modified immunoglobulin locus using Cre-mediatedsite-specific recombination is disclosed. The method involves firsttransfecting an antibody-producing cell with a homology-targeting vectorcomprising a lox site and a targeting sequence homologous to a first DNAsequence adjacent to the region of the immunoglobulin loci of thegenomic sequence which is to be converted to a modified region, so thefirst lox site is inserted into the genomic sequence via site-specifichomologous recombination. Then the cell is transfected with alox-targeting vector comprising a second lox site suitable forCre-mediated recombination with the integrated lox site and a modifyingsequence to convert the region of the immunoglobulin loci to themodified region. This conversion is performed by interacting the loxsites with Cre in vivo, so that the modifying sequence inserts into thegenomic sequence via Cre-mediated site-specific recombination of the loxsites.

Alternatively, monoclonal antibody fragments can be synthesized using anautomated peptide synthesizer, or by expression of full-length gene orof gene fragments in E. coli.

It is further contemplated that monoclonal antibodies may be furtherscreened or optimized for properties relating to specificity, avidity,half-life, immunogenicity, binding association, binding disassociation,or overall functional properties relative to the intended treatment orprotective effect. Thus, it is contemplated that monoclonal antibodiesmay have 1, 2, 3, 4, 5, 6, or more alterations in the amino acidsequence of 1, 2, 3, 4, 5, or 6 CDRs of monoclonal antibodies orhumanized antibodies provided herein. It is contemplated that the aminoacid in position 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of CDR1, CDR2, CDR3,CDR4, CDR5, or CDR6 of the VJ or VDJ region of the light or heavyvariable region of antibodies may have an insertion, deletion, orsubstitution with a conserved or non-conserved amino acid. Such aminoacids that can either be substituted or constitute the substitution aredisclosed above.

In some embodiments, fragments of a whole antibody can perform thefunction of binding antigens. Examples of binding fragments are (i) theFab fragment constituted with the VL, VH, CL and CHI domains; (ii) theFd fragment consisting of the VH and CHI domains; (iii) the Fv fragmentconstituted with the VL and VH domains of a single antibody; (iv) thedAb fragment (Ward, 1989; McCafferty et al., 1990; Holt et al., 2003),which is constituted with a VH or a VL domain; (v) isolated CDR regions;(vi) F(ab′)2 fragments, a bivalent fragment comprising two linked Fabfragments (vii) single chain Fv molecules (scFv), wherein a VH domainand a VL domain are linked by a peptide linker which allows the twodomains to associate to form an antigen binding site (Bird et al., 1988;Huston et al., 1988); (viii) bispecific single chain Fv dimers(PCT/US92/09965) and (ix) “diabodies”, multivalent or multispecificfragments constructed by gene fusion (WO94/13804; Holliger et al.,1993). Fv, scFv or diabody molecules may be stabilized by theincorporation of disulphide bridges linking the VH and VL domains(Reiter et al., 1996). Minibodies comprising a scFv joined to a CH3domain may also be made (Hu et al. 1996). The citations in thisparagraph are all incorporated by reference.

Antibodies also include bispecific antibodies. Bispecific orbifunctional antibodies form a second generation of monoclonalantibodies in which two different variable regions are combined in thesame molecule (Holliger & Winter, 1999). Their use has been demonstratedboth in the diagnostic field and in the therapy field from theircapacity to recruit new effector functions or to target severalmolecules on the surface of tumor cells. Where bispecific antibodies areto be used, these may be conventional bispecific antibodies, which canbe manufactured in a variety of ways (Holliger et al, 1993), e.g.prepared chemically or from hybrid hybridomas, or may be any of thebispecific antibody fragments mentioned above. These antibodies can beobtained by chemical methods (Glennie et al., 1987; Repp et al., 1995)or somatic methods (Staerz & Bevan, 1986) but likewise by geneticengineering techniques which allow the heterodimerization to be forcedand thus facilitate the process of purification of the antibody sought(Merchand et al., 1998). Examples of bispecific antibodies include thoseof the BiTE™ technology in which the binding domains of two antibodieswith different specificity can be used and directly linked via shortflexible peptides. This combines two antibodies on a short singlepolypeptide chain. Diabodies and scFv can be constructed without an Fcregion, using only variable domains, potentially reducing the effects ofanti-idiotypic reaction. The citations in this paragraph are allincorporated by reference.

Bispecific antibodies can be constructed as entire IgG, as bispecificFab′2, as Fab′PEG, as diabodies or else as bispecific scFv. Further, twobispecific antibodies can be linked using routine methods known in theart to form tetravalent antibodies.

Bispecific diabodies, as opposed to bispecific whole antibodies, mayalso be particularly useful because they can be readily constructed andexpressed in E. coli. Diabodies (and many other polypeptides such asantibody fragments) of appropriate binding specificities can be readilyselected using phage display (WO94/13804) from libraries. If one arm ofthe diabody is to be kept constant, for instance, with a specificitydirected against a DC receptor, then a library can be made where theother arm is varied and an antibody of appropriate specificity selected.Bispecific whole antibodies may be made by alternative engineeringmethods as described in Ridgeway et al., 1996), which is herebyincorporated by reference.

Antibody and Polypeptide Conjugates

Embodiments provide antibodies and antibody-like molecules against DCreceptors, polypeptides and peptides that are linked to at least oneagent to form an antibody conjugate or payload. In order to increase theefficacy of antibody molecules as diagnostic or therapeutic agents, itis conventional to link or covalently bind or complex at least onedesired molecule or moiety. Such a molecule or moiety may be, but is notlimited to, at least one effector or reporter molecule. Effectormolecules comprise molecules having a desired activity, e.g., cytotoxicactivity or immunostimulatory activity. Non-limiting examples ofeffector molecules which have been attached to antibodies includeadjuvants, toxins, therapeutic enzymes, antibiotics, radio-labelednucleotides and the like. By contrast, a reporter molecule is defined asany moiety which may be detected using an assay. Non-limiting examplesof reporter molecules which have been conjugated to antibodies includeenzymes, radiolabels, haptens, fluorescent labels, phosphorescentmolecules, chemiluminescent molecules, chromophores, luminescentmolecules, photoaffinity molecules, colored particles or ligands, suchas biotin. The following US patent applications are incorporated byreference to the extent they disclose antibodies, portions ofantibodies, antigens, linkers, specific sequences of such antibodies,antigens and linkers, adjuvants, other components of a fusion protein ortherapeutic composition, host cell or composition, sources of dendriticcells and culturing/activating of dendritic cells and derivatives of andfrom dendritic cells, and methods of use involving such fusion proteins:Ser. Nos. 12/024,036; 12/024,897; 12/025,010; 12/026,095; 12/036,138;12/036,158; 12/504,463; 12/717,778; 12/717,789; 12/717,804; 12/718,365;12/882,052; 12/882,052; 13/100,684; 13/208,993; 13/269,951; 13/282,112;13/415,564; 13/424,582; 13/430,206; 13/594,397; 13/596,526;WO2010/104749; 13/465,371; 13/397,932; PCT/US13/72217; andPCT/US2013/05839.

Certain examples of antibody conjugates are those conjugates in whichthe antibody is linked to a detectable label. “Detectable labels” arecompounds and/or elements that can be detected due to their specificfunctional properties, and/or chemical characteristics, the use of whichallows the antibody to which they are attached to be detected, and/orfurther quantified if desired.

Antibody conjugates include those intended primarily for use in vitro,where the antibody is linked to a secondary binding ligand and/or to anenzyme (an enzyme tag) that will generate a colored product upon contactwith a chromogenic substrate. Examples of suitable enzymes include, butare not limited to, urease, alkaline phosphatase, (horseradish) hydrogenperoxidase or glucose oxidase. Preferred secondary binding ligands arebiotin and/or avidin and streptavidin compounds. The use of such labelsis well known to those of skill in the art and are described, forexample, in U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149 and 4,366,241; each incorporated herein byreference.

Molecules containing azido groups may also be used to form covalentbonds to proteins through reactive nitrene intermediates that aregenerated by low intensity ultraviolet light (Potter & Haley, 1983). Inparticular, 2- and 8-azido analogues of purine nucleotides have beenused as site-directed photoprobes to identify nucleotide bindingproteins in crude cell extracts (Owens & Haley, 1987; Atherton et al.,1985). The 2- and 8-azido nucleotides have also been used to mapnucleotide binding domains of purified proteins (Khatoon et al., 1989;King et al., 1989; and Dholakia et al., 1989) and may be used asantibody binding agents.

Several methods are known in the art for the attachment or conjugationof an antibody to its conjugate moiety. Some attachment methods involvethe use of a metal chelate complex employing, for example, an organicchelating agent such a diethylenetriaminepentaacetic acid anhydride(DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide;and/or tetrachloro-3 -6-diphenylglycouril-3 attached to the antibody(U.S. Pat. Nos. 4,472,509 and 4,938,948, each incorporated herein byreference). Monoclonal antibodies may also be reacted with an enzyme inthe presence of a coupling agent such as glutaraldehyde or periodate.Conjugates with fluorescein markers are prepared in the presence ofthese coupling agents or by reaction with an isothiocyanate. In U.S.Pat. No. 4,938,948 (incorporated herein by reference), imaging of breasttumors is achieved using monoclonal antibodies and the detectableimaging moieties are bound to the antibody using linkers such asmethyl-p-hydroxybenzimidate orN-succinimidyl-3-(4-hydroxyphenyl)propionate.

In some embodiments, derivatization of immunoglobulins by selectivelyintroducing sulfhydryl groups in the Fc region of an immunoglobulin,using reaction conditions that do not alter the antibody combining siteare contemplated. Antibody conjugates produced according to thismethodology are disclosed to exhibit improved longevity, specificity andsensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference).Site-specific attachment of effector or reporter molecules, wherein thereporter or effector molecule is conjugated to a carbohydrate residue inthe Fc region have also been disclosed in the literature (O'Shannessy etal., 1987, incorporated herein by reference). This approach has beenreported to produce diagnostically and therapeutically promisingantibodies which are currently in clinical evaluation.

2. Dendritic Cell Specific Antibodies

In certain aspects, antibodies used to target HPV antigens to dendriticcells are dendritic cell specific antibodies and bind dendritic cellreceptors or receptors expressed by dendritic cells. Some of theantibodies that may be used for this purpose are known in the art.

In some embodiments anti-DCIR antibodies are used to target HPV antigensto dendritic cells. One example includes anti-dendritic cellimmunoreceptor monoclonal antibody conjugates, wherein the conjugatecomprises antigenic peptides that are loaded or chemically coupled tothe antibody. Such antibodies are described in U.S. application No.61/332,465 and are incorporated herein by reference.

In other embodiments anti-CD40 antibodies are used to target HPVantigens to dendritic cells. Compositions and methods for theexpression, secretion and use of anti-CD40 antibodies as vaccines andantigen delivery vectors with one linked antigenic peptides aredescribed in WO 2010/104761; all methods disclosed are incorporatedherein by reference. In some embodiments the anti-CD40 antibodycomprises the heavy chain and light chain variable region frommonoclonal antibody 12E12, 11B6 or 12B4. In other embodiments theanti-CD40 antibody comprises the heavy chain and light chain CDRs frommonoclonal antibody 12E12, 11B6 or 12B4.

In certain aspects anti-LOX-1 antibodies are used to target HPV antigensto dendritic cells. One example of such an antibody can be used totarget the LOX-1 receptor on immune cells and increase the effectivenessof antigen presentation by LOX-1 expressing antigen presenting cells.Examples of such LOX-1 antibodies are described in WO 2008/103953, thecontents of which are incorporated herein by reference.

In other aspects anti-CLEC-6 antibodies are used to target HPV antigensto dendritic cells. One example of such antibodies include anti-CLEC-6antibodies used to increase the effectiveness of antigen presentation byCLEC-6 expressing antigen presenting cells. Such antibodies aredescribed in WO 2008/103947, the methods and contents of which areincorporated herein by reference.

In yet other embodiments anti-Dectin-1 antibodies are used to target HPVantigens to dendritic cells. Anti-Dectin-1 antibodies that increase theeffectiveness of antigen presentation by Dectin-1 expressing antigenpresenting cells are described in WO 2008/118587, the contents of whichare incorporated herein by reference.

In certain aspects, peptide linkers are used to link dendritic cellspecific antibodies and HPV antigens to be presented. Peptide linkersmay incorporate glycosylation sites or introduce secondary structure.Additionally these linkers increase the efficiency of expression orstability of the fusion protein and as a result the efficiency ofantigen presentation to a dendritic cell. Such linkers may includeSSVSPTTSVHPTPTSVPPTPTKSSP (SEQ ID NO: 6); PTSTPADSSTITPTATPTATPTIKG (SEQID NO:29); TVTPTATATPSAIVTTITPTATTKP (SEQ ID NO:30);QTPTNTISVTPTNNSTPTNNSNPKPNP (SEQ ID NO: 5); or TNGSITVAATAPTVTPTVNATPSAA(SEQ ID NO: 31). These examples and others are discussed in WO2010/104747, the contents of which are incorporated herein by reference.

In other embodiments an immune adjuvant is directly fused to thedendritic cell specific antibody in order to enhance the efficacy of thevaccine. In certain aspects the immune adjuvant may be a toll-likereceptor (TLR) agonist. TLR agonists comprise flagellins from Salmonellaenterica or Vibrio cholerae. TLR agonists may be specific for certainTLR classes (i.e., TLR2, TLR5, TLR7 or TLR9 agonists) and may bepresented in any combination or as any modification. Examples of suchimmune adjuvants are described in U.S. application Ser. Nos. 13/208,993,13/415,564, and in WO 2012/021834, the contents of all of which areincorporated herein by reference. US Patent Publications 2012/0039,916and 2012/023,102 are incorporated by reference to the extent theydisclose different TLR agonists.

In some embodiments, the compositions and fusion proteins comprisingdendritic cell antibodies and HPV antigens are used to treat HPV relateddiseases or an HPV related pathology. In some embodiments, an HPVrelated disease is dysplasia, benign neoplasia, pre-malignant neoplasiaor cancer (malignant neoplasia). In some embodiments, the tissue ororgan affected by dysplasia, benign neoplasia, pre-malignant neoplasiaor cancer is the cervix, vulva, vagina, penis, anus, oropharynx, headand neck, throat or lung. In some specific embodiments the HPV relateddiseases or an HPV related pathology is cervical intraepithelialneoplasia (CIN), vulvar intraepithelial neoplasia (VIN), penileintraepithelial neoplasia (PIN), and/or anal intraepithelial neoplasia(AIN). In still other embodiments, the compositions and fusion proteinscomprising dendritic cell antibodies and HPV antigens are used to treatHPV related Common warts, Plantar warts, Flat warts, Anogenital warts,Anal lesions, Genital cancers, Epidermodysplasia verruciformis, Focalepithelial hyperplasia (oral), Oral papillomas, Oropharyngeal cancer,Verrucous cyst or Laryngeal papillomatosis.

III. METHODS OF TREATMENT

As discussed above, the compositions and methods of using thesecompositions can treat a subject (e.g., prevent an HPV infection or HPVrelated disease or evoke a robust or potentiate an immune response toHPV or HPV related disease) having, suspected of having, or at risk ofdeveloping an infection or related disease, related to HPV.

As used herein the phrase “immune response” or its equivalent“immunological response” refers to a humoral (antibody mediated),cellular (mediated by antigen-specific T cells or their secretionproducts) or both humoral and cellular response directed against aprotein, peptide, or polypeptide of the invention in a recipientpatient. Treatment or therapy can be an active immune response inducedby administration of immunogen or a passive therapy effected byadministration of a fusion protein composition, immunogenic compositionor protein composition comprising an antibody/antigen fusion protein,antibody/antigen fusion protein containing material, or primed T-cells.

For purposes of this specification and the accompanying claims the terms“epitope” and “antigenic determinant” are used interchangeably to referto a site on an antigen to which B and/or T cells respond or recognize.B-cell epitopes can be formed both from contiguous amino acids ornoncontiguous amino acids juxtaposed by tertiary folding of a protein.Epitopes formed from contiguous amino acids are typically retained onexposure to denaturing solvents whereas epitopes formed by tertiaryfolding are typically lost on treatment with denaturing solvents. Anepitope typically includes at least 3, and more usually, at least 5 or8-10 amino acids in a unique spatial conformation. Methods ofdetermining spatial conformation of epitopes include those methodsdescribed in Epitope Mapping Protocols (1996). T cells recognizecontinuous epitopes of about nine amino acids for CD8 cells or about13-15 amino acids for CD4 cells. T cells that recognize the epitope canbe identified by in vitro assays that measure antigen-dependentproliferation, as determined by 3H-thymidine incorporation by primed Tcells in response to an epitope (Burke et al., 1994), byantigen-dependent killing (cytotoxic T lymphocyte assay, Tigges et al.,1996, incorporated by reference) or by cytokine secretion.

The presence of a cell-mediated immunological response can be determinedby proliferation assays (CD4 (+) T cells) or CTL (cytotoxic Tlymphocyte) assays. The relative contributions of humoral and cellularresponses to the protective or therapeutic effect of an immunogen can bedistinguished by separately isolating IgG and T-cells from an immunizedsyngeneic animal and measuring protective or therapeutic effect in asecond subject. As used herein and in the claims, the terms “antibody”or “immunoglobulin” are used interchangeably.

Optionally, an antibody or preferably an immunological portion of anantibody, can be chemically conjugated to, or expressed as, a fusionprotein with other proteins. For purposes of this specification and theaccompanying claims, all such fused proteins are included in thedefinition of antibodies or an immunological portion of an antibody.

In one embodiment a method includes treatment for a disease or conditioncaused by or suspected of being caused by an HPV pathogen. In certainaspects embodiments include methods of treatment of HPV infection, suchas an infection acquired from an HPV positive individual. In someembodiments, the treatment is administered in the presence of HPVantigens. Furthermore, in some examples, treatment comprisesadministration of other agents commonly used against viral infection.

The therapeutic compositions are administered in a manner compatiblewith the dosage formulation, and in such amount as will betherapeutically effective. The quantity to be administered depends onthe subject to be treated. Precise amounts of active ingredient requiredto be administered depend on the judgment of the practitioner. Suitableregimes for initial administration and boosters are also variable, butare typified by an initial administration followed by subsequentadministrations.

The manner of application may be varied widely. Any of the conventionalmethods for administration of a polypeptide therapeutic are applicable.These are believed to include oral application on a solidphysiologically acceptable base or in a physiologically acceptabledispersion, parenterally, by injection and the like. The dosage of thecomposition will depend on the route of administration and will varyaccording to the size and health of the subject.

In certain instances, it will be desirable to have multipleadministrations of the composition, e.g., 2, 3, 4, 5, 6 or moreadministrations. The administrations can be at 1, 2, 3, 4, 5, 6, 7, 8,to 5, 6, 7, 8, 9, 10, 11, 12 twelve week intervals, including all rangesthere between.

Combination Therapy

The compositions and related methods, particularly administration of anantibody that binds DC receptor and delivers an HPV antigen or antigensor peptide or peptides to a patient/subject, may also be used incombination with the administration of traditional anti-viral therapiesor anti-cancer therapies or drugs. These include, but are not limitedto, entry inhibitors, CCR5 receptor antagonists, nucleoside reversetranscriptase inhibitors, nucleotide reverse transcriptase inhibitors,non-nucleoside reverse transcriptase inhibitors, protease inhibitors,integrase inhibitors and maturation inhibitors. Anti-cancer therapiesinclude but are not limited to chemotherapy, radiotherapy or radiationtherapy.

The compositions and related methods, particularly administration of anantibody that binds DC receptor and delivers an HPV antigen or antigensor peptide or peptides to a patient/subject, may also be used incombination with the administration of one or more anti-cancer drugsthat include but are not limited to Abiraterone Acetate, Abitrexate(Methotrexate), Abraxane (Paclitaxel Albumin-stabilized NanoparticleFormulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (BrentuximabVedotin), ADE, Ado-Trastuzumab Emtansine, Adriamycin (DoxorubicinHydrochloride), Adrucil (Fluorouracil), Afatinib Dimaleate, Afinitor(Everolimus), Aldara (Imiquimod), Aldesleukin, Alemtuzumab, Alimta(Pemetrexed Disodium), Aloxi (Palonosetron Hydrochloride), Ambochlorin(Chlorambucil), Amboclorin (Chlorambucil), Aminolevulinic Acid,Anastrozole, Aprepitant, Aredia (Pamidronate Disodium), Arimidex(Anastrozole), Aromasin (Exemestane), Arranon (Nelarabine), ArsenicTrioxide, Arzerra (Ofatumumab), Asparaginase Erwinia chrysanthemi,Avastin (Bevacizumab), Axitinib, Azacitidine, BEACOPP, BendamustineHydrochloride, BEP, Bevacizumab, Bexarotene, Bexxar (Tositumomab and I131 Iodine Tositumomab), Bleomycin, Bortezomib, Bosulif (Bosutinib),Bosutinib, Brentuximab Vedotin, Cabazitaxel, Cabozantinib-S-Malate, CAF,Campath (Alemtuzumab), Camptosar (Irinotecan Hydrochloride),Capecitabine, CAPOX, Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, CeeNU(Lomustine), Cerubidine (Daunorubicin Hydrochloride), Cervarix(Recombinant HPV Bivalent Vaccine comprising recombinant L1 protein ofHPV types 16 and 18), Cetuximab, Chlorambucil, CHLORAMBUCIL-PREDNISONE,CHOP, Cisplatin, Clafen (Cyclophosphamide), Clofarabine, Clofarex(Clofarabine), Clolar (Clofarabine), CMF, Cometriq(Cabozantinib-S-Malate), COPP, COPP-ABV, Cosmegen (Dactinomycin),Crizotinib, CVP, Cyclophosphamide, Cyfos (Ifosfamide), Cytarabine,Cytarabine, Liposomal, Cytosar-U (Cytarabine), Cytoxan(Cyclophosphamide), Dabrafenib, Dacarbazine, Dacogen (Decitabine),Dactinomycin, Dasatinib, Daunorubicin Hydrochloride, Decitabine,Degarelix, Denileukin Diftitox, Denosumab, DepoCyt (LiposomalCytarabine), DepoFoam (Liposomal Cytarabine), Dexrazoxane Hydrochloride,Docetaxel, Doxil (Doxorubicin Hydrochloride Liposome), DoxorubicinHydrochloride, Doxorubicin Hydrochloride Liposome, Dox-SL (DoxorubicinHydrochloride Liposome), DTIC-Dome (Dacarbazine), Efudex (Fluorouracil),Elitek (Rasburicase), Ellence (Epirubicin Hydrochloride), Eloxatin(Oxaliplatin), Eltrombopag Olamine, Emend (Aprepitant), Enzalutamide,Epirubicin Hydrochloride, EPOCH, Erbitux (Cetuximab), Eribulin Mesylate,Erivedge (Vismodegib), Erlotinib Hydrochloride, Erwinaze (AsparaginaseErwinia chrysanthemi), Etopophos (Etoposide Phosphate), Etoposide,Etoposide Phosphate, Evacet (Doxorubicin Hydrochloride Liposome),Everolimus, Evista (Raloxifene Hydrochloride), Exemestane, Fareston(Toremifene), Faslodex (Fulvestrant), FEC, Femara (Letrozole),Filgrastim, Fludara (Fludarabine Phosphate), Fludarabine Phosphate,Fluoroplex (Fluorouracil), Fluorouracil, Folex (Methotrexate), Folex PFS(Methotrexate), FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRI-CETUXIMAB,FOLFIRINOX, FOLFOX, Folotyn (Pralatrexate), FU-LV, Fulvestrant, Gardasil(Recombinant HPV Quadrivalent Vaccine comprising recombinant L1 proteinof HPV types 6, 11, 16, and 18), Gazyva (Obinutuzumab), Gefitinib,Gemcitabine Hydrochloride, GEMCITAB INE-CISPLATIN, GEMCITABINE-OXALIPLATIN, Gemtuzumab Ozogamicin, Gemzar (GemcitabineHydrochloride), Gilotrif (Afatinib Dimaleate), Gleevec (ImatinibMesylate), Glucarpidase, Goserelin Acetate, Halaven (Eribulin Mesylate),Herceptin (Trastuzumab), HPV Bivalent Vaccine, Recombinant, HPVQuadrivalent Vaccine, Recombinant, Hycamtin (Topotecan Hydrochloride),Ibritumomab Tiuxetan, Ibrutinib, ICE, Iclusig (Ponatinib Hydrochloride),Ifex (Ifosfamide), Ifosfamide, Ifosfamidum (Ifosfamide), ImatinibMesylate, Imbruvica (Ibrutinib), Imiquimod, Inlyta (Axitinib), Intron A(Recombinant Interferon Alfa-2b), Iodine 131 Tositumomab andTositumomab, Ipilimumab, Iressa (Gefitinib), Irinotecan Hydrochloride,Istodax (Romidepsin), Ixabepilone, Ixempra (Ixabepilone), Jakafi(Ruxolitinib Phosphate), Jevtana (Cabazitaxel), Kadcyla (Ado-TrastuzumabEmtansine), Keoxifene (Raloxifene Hydrochloride), Kepivance(Palifermin), Kyprolis (Carfilzomib), Lapatinib Ditosylate,Lenalidomide, Letrozole, Leucovorin Calcium, Leukeran (Chlorambucil),Leuprolide Acetate, Levulan (Aminolevulinic Acid), Linfolizin(Chlorambucil), LipoDox (Doxorubicin Hydrochloride Liposome), LiposomalCytarabine, Lomustine, Lupron (Leuprolide Acetate), Lupron Depot(Leuprolide Acetate), Lupron Depot-Ped (Leuprolide Acetate), LupronDepot-3 Month (Leuprolide Acetate), Lupron Depot-4 Month (LeuprolideAcetate), Marqibo (Vincristine Sulfate Liposome), Matulane (ProcarbazineHydrochloride), Mechlorethamine Hydrochloride, Megace (MegestrolAcetate), Megestrol Acetate, Mekinist (Trametinib), Mercaptopurine,Mesna, Mesnex (Mesna), Methazolastone (Temozolomide), Methotrexate,Methotrexate LPF (Methotrexate), Mexate (Methotrexate), Mexate-AQ(Methotrexate), Mitomycin C, Mitozytrex (Mitomycin C), MOPP, Mozobil(Plerixafor), Mustargen (Mechlorethamine Hydrochloride), Mutamycin(Mitomycin C), Mylosar (Azacitidine), Mylotarg (Gemtuzumab Ozogamicin),Nanoparticle Paclitaxel (Paclitaxel Albumin-stabilized NanoparticleFormulation), Navelbine (Vinorelbine Tartrate), Nelarabine, Neosar(Cyclophosphamide), Neupogen (Filgrastim), Nexavar (Sorafenib Tosylate),Nilotinib, Nolvadex (Tamoxifen Citrate), Nplate (Romiplostim),Obinutuzumab, Ofatumumab, Omacetaxine Mepesuccinate, Oncaspar(Pegaspargase), Ontak (Denileukin Diftitox), OEPA, OPPA, Oxaliplatin,Paclitaxel, Paclitaxel Albumin-stabilized Nanoparticle Formulation,Palifermin, Palonosetron Hydrochloride, Pamidronate Disodium,Panitumumab, Paraplat (Carboplatin), Paraplatin (Carboplatin), PazopanibHydrochloride, Pegaspargase, Peginterferon Alfa-2b, PEG-Intron(Peginterferon Alfa-2b), Pemetrexed Disodium, Perjeta (Pertuzumab),Pertuzumab, Platinol (Cisplatin), Platinol-AQ (Cisplatin), Plerixafor,Pomalidomide, Pomalyst (Pomalidomide), Ponatinib Hydrochloride,Pralatrexate, Prednisone, Procarbazine Hydrochloride, Proleukin(Aldesleukin), Prolia (Denosumab), Promacta (Eltrombopag Olamine),Provenge (Sipuleucel-T), Purinethol (Mercaptopurine), Radium 223Dichloride, Raloxifene Hydrochloride, Rasburicase, R—CHOP, R—CVP,Recombinant HPV Bivalent Vaccine, Recombinant HPV Quadrivalent Vaccine,Recombinant Interferon Alfa-2b, Regorafenib, Revlimid (Lenalidomide),Rheumatrex (Methotrexate), Rituxan (Rituximab), Rituximab, Romidepsin,Romiplostim, Rubidomycin (Daunorubicin Hydrochloride), RuxolitinibPhosphate, Sclerosol Intrapleural Aerosol (Talc), Sipuleucel-T,Sorafenib Tosylate, Sprycel (Dasatinib), STANFORD V, Sterile Talc Powder(Talc), Steritalc (Talc), Stivarga (Regorafenib), Sunitinib Malate,Sutent (Sunitinib Malate), Sylatron (Peginterferon Alfa-2b), Synovir(Thalidomide), Synribo (Omacetaxine Mepesuccinate), TAC, Tafinlar(Dabrafenib), Talc, Tamoxifen Citrate, Tarabine PFS (Cytarabine),Tarceva (Erlotinib Hydrochloride), Targretin (Bexarotene), Tasigna(Nilotinib), Taxol (Paclitaxel), Taxotere (Docetaxel), Temodar(Temozolomide), Temozolomide, Temsirolimus, Thalidomide, Thalomid(Thalidomide), Toposar (Etoposide), Topotecan Hydrochloride, Toremifene,Torisel (Temsirolimus), Tositumomab and I 131 Iodine Tositumomab, Totect(Dexrazoxane Hydrochloride), Trametinib, Trastuzumab, Treanda(Bendamustine Hydrochloride), Trisenox (Arsenic Trioxide), Tykerb(Lapatinib Ditosylate), Vandetanib, VAMP, Vectibix (Panitumumab), VeIP,Velban (Vinblastine Sulfate), Velcade (Bortezomib), Velsar (VinblastineSulfate), Vemurafenib, VePesid (Etoposide), Viadur (Leuprolide Acetate),Vidaza (Azacitidine), Vinblastine Sulfate, Vincasar PFS (VincristineSulfate), Vincristine Sulfate, Vincristine Sulfate Liposome, VinorelbineTartrate, Vismodegib, Voraxaze (Glucarpidase), Vorinostat, Votrient(Pazopanib Hydrochloride), Wellcovorin (Leucovorin Calcium), Xalkori(Crizotinib), Xeloda (Capecitabine), XELOX, Xgeva (Denosumab), Xofigo(Radium 223 Dichloride), Xtandi (Enzalutamide), Yervoy (Ipilimumab),Zaltrap (Ziv-Aflibercept), Zelboraf (Vemurafenib), Zevalin (IbritumomabTiuxetan), Zinecard (Dexrazoxane Hydrochloride), Ziv-Aflibercept,Zoladex (Goserelin Acetate), Zoledronic Acid, Zolinza (Vorinostat),Zometa (Zoledronic Acid) and Zytiga (Abiraterone Acetate).

The compositions and related methods, particularly administration of anantibody that binds DC receptor and delivers an HPV antigen or antigensor peptide or peptides to a patient/subject, may also be used incombination with the administration of radiation therapy that includesbut is not limited to X-rays, gamma rays, and charged particles. Theradiation may be delivered by a machine outside the body (external-beamradiation therapy), or it may come from radioactive material placed inthe body near cancer cells (internal radiation therapy orbrachytherapy). Internal radiation therapy may be systemic (e.g.radioactive iodine). External-beam radiation therapy may include, but isnot limited to, 3-dimensional conformal radiation therapy (3D-CRT),Intensity-modulated radiation therapy (IMRT), Image-guided radiationtherapy (IGRT), Tomotherapy, Stereotactic radiosurgery (SRS),Stereotactic body radiation therapy (SBRT), Proton therapy or othercharged particle beams (e.g., electron beams). Internal radiationtherapy or brachytherapy may comprise interstitial brachytherapy whichuses a radiation source placed within tumor tissue and may be used todeliver a dose higher than external beam radiation while causing lessdamage to normal tissue. Brachytherapy may be given as a low-dose rateor high-dose rate treatment. In additional embodiments, brachytherapymay be permanent or temporary. Radiation therapy may comprise systemicradiation therapy. Systemic radiation therapy may comprise a swallowedor injected radioactive substance, that includes, but is not limited toany single, multiple or combination dose of Radioactive iodine (¹³¹I),ibritumomab tiuxetan (Zevalin®), 131 tositumomab (Bexxar®),samarium-153-lexidronam (Quadramet®) and strontium-89 chloride(Metastron®) or any monoclonal bound to a radioactive substance. Thedose of radiation according to different embodiments may be tailored tothe specific disease, condition or cancered being treated. In someembodiments, the single or total dose may be 1-10 gray(Gy), 10-20 Gy,20-40 Gy, 40-60 Gy, or 60-80 Gy, or any value or rage derivable therein.In some embodiments, radiation therapy or dose may be fractionated. Inone embodiment, a total dose may be fractionated per day or per week. Incertain embodiments the daily fractionated dose may be 1.8-2 Gy. It iscontemplated that a total dose may be fractionated into daily or weeklydoses in the range of 0.1 Gy to 10 Gy.

In one aspect, it is contemplated that a therapy is used in conjunctionwith antiviral or anti-cancer therapies. Alternatively, the therapy mayprecede or follow the other agent treatment by intervals ranging fromminutes to weeks. In embodiments where the other agents and/or aproteins or polynucleotides are administered separately, one wouldgenerally ensure that a significant period of time did not expirebetween the time of each delivery, such that the therapeutic compositionwould still be able to exert an advantageously combined effect on thesubject. In such instances, it is contemplated that one may administerboth modalities within about 12-24 h of each other and, more preferably,within about 6-12 h of each other. In some situations, it may bedesirable to extend the time period for administration significantly,however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2,3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

In yet another aspect, a vaccine may be administered as part of aprime/boost strategy. A priming vaccine dose can be administered using aDC specific antibody fused to an HPV antigen in any of the embodimentsdescribed herein. A vaccine boost can be administered through the use ofa second vaccine, either of the same type or from a different type ofvaccine. Examples of a separate HPV vaccine include Gardasil™(recombinant HPV quadrivalent vaccine comprising recombinant L1 proteinof HPV types 6, 11, 16, and 18) or Cervarix™ (recombinant HPV bivalentvaccine comprising recombinant L1 protein of HPV types 16 and 18).Additional examples of such different vaccines include naked DNAvaccines or a recombinant viruses. The second vaccine may compriseadditional HPV antigens apart from the E6 or E7 antigens that may beused in the first vaccine. It is also contemplated that the secondvaccine may comprise an HPV protein such as an E6 or E7 protein plus anadjuvant either directly linked or administered independently.

Various combinations of therapy may be employed, for example antiviralor anti-cancer therapy is “A” and an antibody vaccine that comprises anantibody that binds a DC receptor and delivers an HPV antigen or apeptide or consensus peptide thereof is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/A

Administration of the antibody compositions to a patient/subject willfollow general protocols for the administration of such compounds,taking into account the toxicity, if any, of the composition. It isexpected that the treatment cycles would be repeated as necessary. It isalso contemplated that various standard therapies, such as hydration,may be applied in combination with the described therapy.

General Pharmaceutical Compositions

In some embodiments, pharmaceutical compositions are administered to asubject. Different aspects may involve administering an effective amountof a composition to a subject. In some embodiments, an antibody thatbinds DC receptor and delivers an HPV antigen or a peptide or consensuspeptide thereof may be administered to the patient to protect against ortreat infection by one or more HPV types or protect or treat against oneor more HPV related diseases such as cancer. Alternatively, anexpression vector encoding one or more such antibodies or polypeptidesor peptides may be given to a patient as a preventative treatment.Additionally, such compositions can be administered in combination withan antibiotic, antiviral or anticancer agent. Such compositions willgenerally be dissolved or dispersed in a pharmaceutically acceptablecarrier or aqueous medium.

The phrases “pharmaceutically acceptable” or “pharmacologicallyacceptable” refer to molecular entities and compositions that do notproduce an adverse, allergic, or other untoward reaction whenadministered to an animal or human. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like. The use of such media and agents forpharmaceutical active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredients, its use in immunogenic and therapeutic compositionsis contemplated. Supplementary active ingredients, such as otheranti-infective agents and vaccines, can also be incorporated into thecompositions.

The active compounds can be formulated for parenteral administration,e.g., formulated for injection via the mucosal, intravenous,intramuscular, sub-cutaneous, intratumoral or even intraperitonealroutes. Typically, such compositions can be prepared as either liquidsolutions or suspensions; solid forms suitable for use to preparesolutions or suspensions upon the addition of a liquid prior toinjection can also be prepared; and, the preparations can also beemulsified.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil, or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that it may be easily injected. It also should be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi.

The proteinaceous compositions may be formulated into a neutral or saltform. Pharmaceutically acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like.

A pharmaceutical composition can include a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating, such as lecithin,by the maintenance of the required particle size in the case ofdispersion, and by the use of surfactants. The prevention of the actionof microorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization or an equivalent procedure. Generally,dispersions are prepared by incorporating the various sterilized activeingredients into a sterile vehicle which contains the basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum-drying andfreeze-drying techniques, which yield a powder of the active ingredient,plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Administration of the compositions will typically be via any commonroute. This includes, but is not limited to oral, nasal, or buccaladministration. Alternatively, administration may be by orthotopic,intradermal, subcutaneous, intramuscular, intraperitoneal, intratumoral,intranasal, or intravenous injection. In certain embodiments, a vaccinecomposition may be inhaled (e.g., U.S. Pat. No. 6,651,655, which isspecifically incorporated by reference). Such compositions wouldnormally be administered as pharmaceutically acceptable compositionsthat include physiologically acceptable carriers, buffers or otherexcipients.

An effective amount of therapeutic or prophylactic composition isdetermined based on the intended goal. The term “unit dose” or “dosage”refers to physically discrete units suitable for use in a subject, eachunit containing a predetermined quantity of the composition calculatedto produce the desired responses discussed above in association with itsadministration, i.e., the appropriate route and regimen. The quantity tobe administered, both according to number of treatments and unit dose,depends on the protection desired.

Precise amounts of the composition also depend on the judgment of thepractitioner and are peculiar to each individual. Factors affecting doseinclude physical and clinical state of the subject, route ofadministration, intended goal of treatment (alleviation of symptomsversus cure), and potency, stability, and toxicity of the particularcomposition.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeutically orprophylactically effective. The formulations are easily administered ina variety of dosage forms, such as the type of injectable solutionsdescribed above.

IV. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1—Recombinant Fusion Proteins of Anti-DC Receptors (DCRs) andHPV E6 and E6 Fusion Proteins

The inventors' scheme for the development of expression constructs forproduction of anti-DC receptor antibodies fused to E6 and E7 sequencesfrom HPV 16 and 18 is given in FIG. 1. The scheme identifies an order ofantigen cassettes encoding E6 and E7 from HPV 16 and 18 that isefficiently secreted and are intact when fused to the H chainC-terminus. There are 64 possible combinations of just these 4sequences, and very many more when interspersed with flexible linkersequences. The inventors' strategy is a stepwise approach starting witheach antigen alone, with and without a preceding flexible linker [8initial constructs], then selecting those vectors that express mostefficiently for adding on additional cassettes. Each cycle ofconstruction and testing takes one week. Establishing the finalproduction CHO-S cell lines take a further 8 weeks, including scale-upto levels suitable for vaccine production to preclinical studies inhuman in vitro and animal in vivo.

A transient expression vector encoding the antibody heavy chain has anin-frame Nhe I site at the C-terminus and antigen or flexible linkerencoding Spe I-Not I cassettes are inserted between the vector Nhe I andNot I sites. The vector Nhe I site is lost in this ligation, but eachcassette encodes a new C-terminal in-frame Nhe I site. Thus, additionalantigen or linker cassettes can be added in an iterative fashion. Eachnew construct is transiently transfected into 293F cells with a matchinglight chain vector and at 72 hr secreted vaccine is isolated by proteinA affinity and analyzed by SDS.PAGE. Constructs that express well arethe preferred vectors for adding new cassettes (FIG. 1).

The inventors have engineered expression constructs with HPV16 E6 and E7and HPV18 E6 and E7 sequences fused to antibody heavy chain C-terminii.Constructs with HPV16 or HPV18 E6 or HPV16 or HPV18 E7 sequences fuseddirectly to an anti-dendritic cell receptor antibody heavy chainC-terminal codon failed to be secrete any detectable vaccine whenco-transfected into 293F cells with a matching light chain expressionvector (not shown). However, similar vectors incorporating a flexiblelinker sequence (Flex v1), secreted the vaccines (FIG. 2, lanes 1 and3). Furthermore, constructs adding HPV E6 onto the Flex v1 HPV E7 vectorand vice versa, also secreted vaccine (FIG. 2, lanes 2 and 4).Successful expression of such vaccines is independent on the variableregion sequences. Thus, the Flex v1-HPV E6/E7 sequence was transferredto established vectors for stable expression of anti-CD40-Flex v1-HPV16or 18 E6-HPV16E7, anti-Langerin-Flex v1-HPV16 or 18 E6-HPV16E7 andcontrol hIgG4-Flex-v1-HPV16 or 18 E6-HPV16E7 in CHO-S cells.

Anti-CD40-HPV16.E6/7 can efficiently bind to DCs in peripheral blood ofhealthy donors. Peripheral blood mononuclear cells (PBMCs) were acquiredfrom the blood of healthy individuals. PBMCs were incubated for 15 minon ice in the presence of different amounts of anti-CD40-HPV16.E6/7 orcontrol IgG4-HPV16.E6/7 proteins. Cells were washed vigorously and thenstained with anti-E6/7 antibodies to detect cell surface bound proteins.As shown in FIG. 3, anti-CD40-HPV16.E6/7 can efficiently bind to humanblood DCs (CD3-CD19-CD14-HLA-DR+CD11c+). In contrast, IgG4-HPV16.E6/7did not bind to the same DCs. Anti-CD40-HPV18.E6/7 also bound to DCs inperipheral blood of healthy donors (data not shown).

Anti-CD40-HPV16.E6/7 can efficiently activate E6/7-specific memory CD4+and CD8+ T cells from HPV-related cancer patients. Evaluated next wasthe in vitro immunogenicity of anti-CD40-HPV16.E6/7 using PBMCs fromHPV-positive head and neck cancer patients. Patient PBMCs were loadedwith recombinant HPV16.E6/7 proteins, anti-CD40-HPV16.E6/7, or peptidepool of HPV16.E6/7 proteins. In this experiment, the same molarconcentration of E6/7 in each protein was applied to compare the levelsof E6/7-specific IFNg-expressing CD4+ and CD8+ T cell responses. After 7days in vitro culture, PBMCs were restimulated for 5 h with peptide poolof E6/7 in the presence of brefeldin A and then cells were stained forIFNg expression. FIG. 4 shows that anti-CD40-HPV16.E6/7 was moreefficient than HPV16.E6/7 at eliciting IFNg+CD4+ and IFNg+CD8+ T cellresponses. The levels of HPV16.E6/7-specific IFNg+CD4+ T cell responseselicited with anti-CD40-HPV16.E6/7 was similar to those elicited by thepeptide pool that was used as a positive control. Thus, our new vaccinemodels composed of anti-DCR and HPV antigens, including E6/7, is highlyeffective in activating antigen-specific cellular immune responses inthe patients who have HPV-related cancers. Furthermore, such HPV antigen(E6 and E7)-specific CD8+ CTLs are expected to efficiently suppresstumor progression and could result in the rejection of tumors inpatients.

Anti-CD40-HPV16.E6/7 can prime E6/7-specific CD4+ and CD8+ T cellresponses in vivo. To test the in vivo immunogenicity ofanti-CD40-HPV16.E6/7 vaccine, human CD40 transgenic mice were used. Fiveanimals were immunized s.c. with 30 ug anti-CD40-HPV16.E6/7 plus poly ICon day 0 and then boosted twice with the same vaccine. On day 7 afterthe second boosting, CD4+ and CD8+ T cells were purified from spleensand then restimulated with oen of HPV16.E6/7 peptide clusters 1-5, none,a peptide pool of prostate specific antigen (PSA), or a HPV16.E6/7peptide pool. FIG. 5 shows that anti-CD40-HPV16.E6/7 induce HPV16 E6/7peptide clusters 2 and 3-specific CD4+ and cluster 5-specific CD8+ Tcell responses in the human CD40 transgenic mice. Importantly, thelevels of E6/7-specific CD8+ T cell responses were greater than thelevels of E6/7-specific CD4+ T cell responses. This indicates thatanti-CD40-HPV16.E6/7 vaccines are particularly efficient in elicitingCD8+ CTLs that can kill HPV-infected cells and tumor cells. Each dot inFIG. 5 represent the data generated with a single mouse.

Anti-CD40-HPV16.E6/7 can suppress TC-1 tumor progression in the humanCD40 transgenic mice. Efficacy of anti-CD40-HPV16.E6/7 plus poly ICvaccine was tested in TC-1 challenged human CD40 transgenic mice. Twogroups of animals (Human CD40+ and human CD40− mice, 5 mice per group)were challenged on day 0 with TC-1 tumor cell line subcutaneously. Ondays 6 and 12, animals were immunized with anti-CD40-HPV.E6/7 plus polyIC. Tumor progression was assessed and presented in FIG. 6. By day 14after TC-1 challenge, both human CD40+ and CD40− mice developed similarsizes of tumors. In the human CD40− animals TC-1 tumor progressedquickly and reached 1000 mm³ on day 25 after the challenge. However,TC-1 tumor progression in the human CD40+ mice was significantlydelayed. Our data demonstrate that anti-CD40-HPV16.E6/7 vaccine targetshuman CD40 and thus elicits E6/7-specific CD8+ CTLs, as shown in FIG. 5,that suppress TC-1 tumor progression in the animals.

The effects of poly IC, poly IC plus montanide, GM-CSF plus montanide,and montanide alone on the immunogenicity of anti-CD40-HPV.E6/7 vaccinewas tested in the human CD40 transgenic mice. Four animals in each groupwere immunized s.c. with 30 ug anti-CD40-HPV.E6/7 alone oranti-CD40-HPV16.E6/7 with indicated adjuvants (FIG. 7). After 7 days,blood from individual animals were harvested and stained with tetramer.As shown in the figure below, poly IC was able to effectively promoteanti-CD40-HPV16.E6/7-specific CD8+ T cell responses. Montanide alone orGM-CSF in montanide did not significantly promote E6/7-specific CD8+ Tcell responses.

Sequences below are based on the humanized 12E12 anti-human CD40 VK2 VH2antibody-protein sequences are the expected mature secreted proteinsequence and the DNA sequences include the initiator ATG and the leaderpeptide region. Alternately, the HPV18 sequences can be grafted onto theC-terminus of the VK2 chain and a broader spectrum vaccine produced bycombining this with the HPV16 sequences on the VH2 H chain.

HPV 16 E6 see below SEQ ID NO: 1 HPV 16 E7 see below SEQ ID NO: 2HPV 18 E6 see below SEQ ID NO: 3 HPV 18 E7 see below SEQ ID NO: 4 Flexv1see below SEQ ID NO: 5 f1 see below SEQ ID NO: 6hAnti-CD40VK2-LV-hIgGK-C see below SEQ ID NO: 7 hAnti-CD40VH2-LV-hIgG4H-see below SEQ ID NO: 8 C Anti-CD4012E12 light chain see belowSEQ ID NO: 9 variable region Anti-CD4012E12 heavy chain see belowSEQ ID NO: 10 variable region Anti-CD40 12E12 CDR1L SASQGISNYLNSEQ ID NO: 11 Anti-CD40 12E12 CDR2L YTSILHS SEQ ID NO: 12Anti-CD40 12E12 CDR3L QQFNKLPPT SEQ ID NO: 13 Anti-CD40 12E12 CDR1HGFTFSDYYMY SEQ ID NO: 14 Anti-CD40 12E12 CDR2H YINSGGGSTYYPDTVKGSEQ ID NO: 15 Anti-CD40 12E12 CDR3H RGLPFHAMDY SEQ ID NO: 16 HPV 16 E6MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVGDFAFRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSVYGTTLEQQYNKPLCDLLIRCINCQ KPLCPE(SEQ ID NO: 1) HPV 16 E7MHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTF CCK(SEQ ID NO: 2) HPV 18 E6MARFEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVGEFAFKDLFVVYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLRCQKPLNP (SEQ ID NO: 3)HPV 18 E7 MHGPKATLQDIVLHLEPQNEIPVDLLGHGQLSDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCCK (SEQ ID NO: 4) Flexv1 QTPTNTISVTPTNNSTPTNNSNPKPNP(SEQ ID NO: 5) f1 SSVSPTTSVHPTPTSVPPTPTKSSP (SEQ ID NO: 6)hAnti-CD40VK2-LV-hIgGK-CDIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKAVKLLIYYTSILHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQFNKLPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 7)hAnti-CD40VH2-LV-hIgG4H-CEVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQAPGKGLEWVAYINSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARRGLPFHAMDYWGQGTLVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 8)Anti-CD4012E12 light chain variable regionDIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSILHSGVPSRFSGSGSGTDYSLTIGNLEPEDIATYYCQQFNKLPPTFGGGTKLEIK (SEQ ID NO: 9)Anti-CD4012E12 heavy chain variable regionCEVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQTPEKRLEWVAYINSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARRGLPFHAMDYWG QGTSVTVS(SEQ ID NO: 10) hAnti-CD40VK2-LV-hIgGK-CDIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKAVKLLIYYTSILHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQFNKLPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 17)hAnti-CD40VK2-LV-hIgGK-C DNA sequence (includes the leader peptideregion) ATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCCAGGCGCGCGATGTGATATCCAGATGACACAGAGCCCTTCCTCCCTGTCTGCCTCTGTGGGAGACAGAGTCACCATCACCTGCAGTGCAAGTCAGGGCATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGGCAAGGCCGTTAAACTCCTGATCTATTACACATCAATTTTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGACAGATTATACCCTCACCATCAGCTCCCTGCAGCCTGAAGATTTCGCCACTTACTATTGTCAGCAGTTTAATAAGCTTCCTCCGACGTTCGGTGGAGGCACCAAACTCGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTATGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 18)hAnti-CD40VH2-LV-hIgG4H-C-Flex-v1-HPV16-E6-HPV16-E7-f1(Bold, italicized single underline sequence is HPV16 E6; bold,italicized double underline sequence is HPV16 E7; non-bolded, non-italicized single underlined (Flexv1) and non-bolded, non-italicizeddouble underlined (f1) sequences are flexible glycosylated linkersequences) EVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQAPGKGLEWVAYINSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARRGLPFHAMDYWGQGTLVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQTPTNTISVTPTNNSTPTNNSNPKPNPAS

 

AS

ASSSVSPTTSVHPTPTSVPPTPTKS SPAS (SEQ ID NO: 19)hAnti-CD40VH2-LV-hIgG4H-C-Flex-v1-HPV16-E6-HPV16-E7-f1 DNA sequence(includes the leader peptide region)ATGGGTTGGAGCCTCATCTTGCTCTTCCTTGTCGCTGTTGCTACGCGTGTCCACTCCGAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCCGGAGGGTCCCTGAAACTCTCCTGTGCAACCTCTGGATTCACTTTCAGTGACTATTACATGTATTGGGTTCGCCAGGCCCCAGGCAAGGGCCTGGAGTGGGTCGCATACATTAATTCTGGTGGTGGTAGCACCTATTATCCAGACACTGTAAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAACAGCCTGAGGGCCGAGGACACAGCCGTGTATTACTGTGCAAGACGGGGGTTACCGTTCCATGCTATGGACTATTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCAGCCAAAACGAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCGAAGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAGTCAGACCCCCACCAACACCATCAGCGTGACCCCCACCAACAACAGCACCCCCACCAACAACAGCAACCCCAAGCCCAACCCCGCTAGTATGCACCAAAAAAGGACCGCAATGTTTCAGGACCCCCAAGAGAGGCCCCGCAAACTGCCACAACTTTGCACGGAGCTGCAGACAACAATACATGACATCATTCTCGAATGTGTTTACTGTAAGCAGCAGTTGTTGCGAAGAGAAGTGGGAGACTTCGCTTTCAGAGACCTGTGTATCGTATATCGCGATGGCAATCCTTATGCCGTCTGCGATAAATGCCTCAAGTTTTACTCCAAGATCAGCGAGTACCGGCACTACTGTTACTCTGTGTATGGGACTACCCTCGAACAGCAGTATAACAAGCCGCTGTGCGATCTCCTTATCCGGTGCATTAACTGCCAGAAGCCACTGTGTCCTGAGGCTAGTATGCACGGGGATACCCCCACACTCCACGAATACATGCTTGATTTGCAACCTGAAACGACCGACCTGTACGGCTATGGTCAGCTGAATGACTCCAGCGAGGAAGAGGATGAGATTGACGGACCGGCAGGCCAGGCCGAGCCAGACCGGGCTCATTATAACATCGTGACTTTCTGCTGTAAGGCTAGTAGCAGCGTGAGCCCCACCACCAGCGTGCACCCCACCCCCACCAGCGTGCCCCCCACCCCCACCAAGAGCAGCCCCGCTAGCTGA (SEQ ID NO: 20)hAnti-CD40VH2-LV-hIgG4H-C-Flex-v1-HPV18E6-HPV18E7-f1(Bold, italicized single underline sequence is HPV18 E6; bold,italicized double underline sequence is HPV18 E7; non-bolded, non-italicized single underlined (Flexv1) and non-bolded, non-italicizeddouble underlined (f1) sequences are flexible glycosylated linkersequences) EVKLVESGGGLVQPGGSLKLSCATSGFTFSDYYMYWVRQAPGKGLEWVAYINSGGGSTYYPDTVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARRGLPFHAMDYWGQGTLVTVSSAKTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKASQTPTNTISVTPTNNSTPTNNSNPKPNPAS

AS

ASSSVSPTTSVHPTPTSVPPTPTK SSPAS (SEQ ID NO: 21)hAnti-CD40VH2-LV-hIgG4H-C-Flex-v1-HPV18E6-HPV18E7-f1 DNA sequence(includes the leader peptide region)ATGGGTTGGAGCCTCATCTTGCTCTTCCTTGTCGCTGTTGCTACGCGTGTCCACTCCGAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCCGGAGGGTCCCTGAAACTCTCCTGTGCAACCTCTGGATTCACTTTCAGTGACTATTACATGTATTGGGTTCGCCAGGCCCCAGGCAAGGGCCTGGAGTGGGTCGCATACATTAATTCTGGTGGTGGTAGCACCTATTATCCAGACACTGTAAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAACAGCCTGAGGGCCGAGGACACAGCCGTGTATTACTGTGCAAGACGGGGGTTACCGTTCCATGCTATGGACTATTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCAGCCAAAACGAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCGAAGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAGTCAGACCCCCACCAACACCATCAGCGTGACCCCCACCAACAACAGCACCCCCACCAACAACAGCAACCCCAAGCCCAACCCCGCTAGTATGGCCAGATTCGAGGATCCAACACGCCGACCTTACAAATTGCCGGACCTTTGCACGGAGCTGAACACTTCCCTGCAGGACATAGAAATTACCTGCGTCTACTGCAAGACCGTTCTCGAACTGACAGAAGTAGGCGAGTTTGCGTTTAAAGATCTGTTCGTGGTGTATCGGGATAGCATTCCCCACGCAGCTTGTCATAAGTGTATCGACTTCTATTCTAGGATCCGGGAGCTCAGACACTATAGCGATTCCGTGTACGGCGACACACTTGAGAAGCTCACTAACACCGGGCTGTACAACCTCCTGATCCGGTGCTTGAGGTGTCAGAAACCCCTGAATCCTGCTAGTATGCACGGGCCTAAGGCCACACTGCAAGATATTGTCCTCCATCTCGAACCCCAGAATGAGATACCAGTGGACCTTCTGGGCCACGGACAGTTGTCCGATAGCGAGGAGGAAAACGACGAAATCGACGGTGTTAACCACCAGCACTTGCCGGCTCGGAGGGCAGAGCCCCAGAGACATACCATGCTGTGCATGTGTTGCAAAGCTAGTAGCAGCGTGAGCCCCACCACCAGCGTGCACCCCACCCCCACCAGCGTGCCCCCCACCCCCACCAAGAGCAGCCCCGCTAGCTGA (SEQ ID NO: 22)

Example 2—Recombinant Fusion Proteins of Anti-DC Receptors (DCRs), TLRLigands, and HPV Sequences

Below is an example of a TLR2 ligand (tri-acylated cohesin, expressed inE. coli) where the C residue in the D1 leader domain is lipidated, thiscan be non-covalently attached to anti-CD40-HPV vaccine when theanti-CD40 has, e.g., a Dockerin domain fused to either the C-terminus orthe L chain or the H chain C-terminus distal to the HPV E6/7 sequences.

D1-6His-Cohesin-Nhe-Spe-Not (note that additionalcancer antigen sequences can be added distal to the Cohesin domain)(SEQ ID NO: 23) MKKLLIAAMMAAALAACSQEAKQEVKEAVQAVESDVKDTAMGSSHHHHHHSSGLVPRGSHMASMDLDAVRIKVDTVNAKPGDTVNIPVRFSGIPSKGIANCDFVYSYDPNVLEIIEIKPGELIVDPNPTKSFDTAVYPDRKMIVFLFAEDSGTGAYAITKDGVFATIVAKVKEGAPNGLSVIKFVEVGGFANNDLVEQKTQFFDGGVNVGDTTEPATPTTPVTTPTTTDDLDAASLIKTSEFD1-6His-Cohesin-Nhe-Spe-Not DNA sequence (SEQ ID NO: 24)ATGAAAAAACTGCTGATTGCCGCCATGATGGCTGCAGCTCTGGCCGCATGCAGCCAGGAAGCCAAACAGGAAGTGAAAGAAGCCGTGCAGGCCGTGGAAAGCGATGTGAAAGATACCGCCATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGTATGGATCTGGATGCAGTAAGGATTAAAGTGGACACAGTAAATGCAAAACCGGGAGACACAGTAAATATACCTGTAAGATTCAGTGGTATACCATCCAAGGGAATAGCAAACTGTGACTTTGTATACAGCTATGACCCGAATGTACTTGAGATAATAGAGATAAAACCGGGAGAATTGATAGTTGACCCGAATCCTACCAAGAGCTTTGATACTGCAGTATATCCTGACAGAAAGATGATAGTATTCCTGTTTGCGGAAGACAGCGGAACAGGAGCGTATGCAATAACTAAAGACGGAGTATTTGCTACGATAGTAGCGAAAGTAAAAGAAGGAGCACCTAACGGGCTCAGTGTAATCAAATTTGTAGAAGTAGGCGGATTTGCGAACAATGACCTTGTAGAACAGAAGACACAGTTCTTTGACGGTGGAGTAAATGTTGGAGATACAACAGAACCTGCAACACCTACAACACCTGTAACAACACCGACAACAACAGATGATCTAGATGCAGCTAGCTTAATTAAAACTAGTGAATTCTGA

Below is an example of an anti-CD40 L chain bearing a preferred TLR5LFlagellin domain (shown underlined). This would be co-transfected withthe matching H chain bearing HPV E6/7 antigen at the C-terminus.

hAnti-CD40VK2-LV-hIgGK-C-Flgn1-Flgn2(underlined sequence is Flagellin domain) (SEQ ID NO: 25)DIQMTQSPSSLSASVGDRVTITCSASQGISNYLNWYQQKPGKAVKLLIYYTSILHSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQFNKLPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECASIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQASQPELAEAAAKTTENPLQKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRAQILQAShAnti-CD40VK2-LV-hIgGK-C-Flgn1-Flgn2 DNA sequence (SEQ ID NO: 26)ATGAGGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGCTCCCAGGCGCGCGATGTGATATCCAGATGACACAGAGCCCTTCCTCCCTGTCTGCCTCTGTGGGAGACAGAGTCACCATCACCTGCAGTGCAAGTCAGGGCATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGGCAAGGCCGTTAAACTCCTGATCTATTACACATCAATTTTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGACAGATTATACCCTCACCATCAGCTCCCTGCAGCCTGAAGATTTCGCCACTTACTATTGTCAGCAGTTTAATAAGCTTCCTCCGACGTTCGGTGGAGGCACCAAACTCGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTATGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGCTAGTATCGAGCGTCTGTCTTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGTCAGGCGATTGCTAACCGTTTTACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATCGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCTAACAGCACTAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGTCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGTGCCAACGACGGTGAAACTATCGATATCGATCTGAAGCAGATCAACTCTCAGACCCTGGGCCTGGATTCACTGAACGTGCAGGCTAGTCAACCAGAGCTGGCGGAAGCAGCCGCTAAAACCACCGAAAACCCGCTGCAGAAAATTGATGCCGCGCTGGCGCAGGTGGATGCGCTGCGCTCTGATCTGGGTGCGGTACAAAACCGTTTCAACTCCGCTATCACCAACTTGGGCAATACCGTAAACAACCTGTCTGAAGCGCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCGCAGATTCTGCAGGC TAGCTGA

Below is an example of analogous HPV 18 E6/7 sequences fused toDC-targeting antibody H chain (in this case anti-Langerin). This can befused instead to the H chain of the preferred antiCD40 antibody in placeof the HPV 16 sequences, or fused downstream of the HPV 16 sequences, orfused to the anti-CD40 L chain—in each case making a vaccine bearingboth HPV 16 and HPV 18 sequences.

Anti-Langerin15B10H-LV-hIgG4H-C-HPV18E7-HPV18E6-f1(Bold, italicized single underline sequence isHPV18 E6; bold, italicized double underlinesequence is HPV18 E7; non-bolded, non-italicizeddouble underlined (f1) sequence is a flexibleglycosylated linker sequence) (SEQ ID NO: 27)QVQLRQSGPELVKPGASVKMSCKASGYTFTDYVISWVKQRTGQGLEWIGDIYPGSGYSFYNENFKGKATLTADKSSTTAYMQLSSLTSEDSAVYFCATYYNYPFAYWGQGTLVTVSAAKTTGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKAS

AS

orASSSVSPTTSVHPTPTS VPPTPTKSSPASAnti-Langerin15B10H-LV-hIgG4H-C-HPV18E7-HPV18E6-f1 DNA sequence(SEQ ID NO: 28) ATGGAATGGAGGATCTTTCTCTTCATCCTGTCAGGAACTGCAGGTGTCCACTCCCAGGTTCAGCTGCGGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTTACTGACTATGTTATAAGTTGGGTGAAGCAGAGAACTGGACAGGGCCTTGAGTGGATTGGAGATATTTATCCTGGAAGTGGTTATTCTTTCTACAATGAGAACTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCACCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGTGCAACCTACTATAACTACCCTTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACAACGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGTTCGAAGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAAGCTAGTATGCACGGGCCTAAGGCCACACTGCAAGATATTGTCCTCCATCTCGAACCCCAGAATGAGATACCAGTGGACCTTCTGGGCCACGGACAGTTGTCCGATAGCGAGGAGGAAAACGACGAAATCGACGGTGTTAACCACCAGCACTTGCCGGCTCGGAGGGCAGAGCCCCAGAGACATACCATGCTGTGCATGTGTTGCAAAGCTAGTATGGCCAGATTCGAGGATCCAACACGCCGACCTTACAAATTGCCGGACCTTTGCACGGAGCTGAACACTTCCCTGCAGGACATAGAAATTACCTGCGTCTACTGCAAGACCGTTCTCGAACTGACAGAAGTAGGCGAGTTTGCGTTTAAAGATCTGTTCGTGGTGTATCGGGATAGCATTCCCCACGCAGCTTGTCATAAGTGTATCGACTTCTATTCTAGGATCCGGGAGCTCAGACACTATAGCGATTCCGTGTACGGCGACACACTTGAGAAGCTCACTAACACCGGGCTGTACAACCTCCTGATCCGGTGCTTGAGGTGTCAGAAACCCCTGAATCCTGCTAGTAGCAGCGTGAGCCCCACCACCAGCGTGCACCCCACCCCCACCAGCGTGCCCCCCACCCCCACCAAGAGCAGCCCCGCTAGCTGA

Example 3—CD40 Targeting HPV Vaccine (CD40HVac)

CD40HVac plus poly IC induces E6/7-specific CD8+ CTLs in human CD40transgenic B6 (hCD40Tg) mice. hCD40Tg and WT animals (5 mice/group) wereimmunized subcutaneously (SC) with 30 μg CD40HVac plus 50 μg poly IC inPBS (100 μl) and boosted twice two weeks apart. The amounts of CD40HVacand poly IC were predetermined in separate experiments. Seven days afterthe second boosting, IFNγ ELISPOT was performed using purified CD8+ Tcells from spleens (FIG. 8a ). Compared to WT mice, hCD40Tg miceelicited increased numbers of CD8+IFNγ+ T cells. The inventors alsoobserved that hCD40Tg mice had increased E7-specific CD8+ T cells in theblood, as measured by tetramer staining (FIG. 8b ). In addition,CD40HVac plus poly IC induced greater levels of E6/7-specific CD4+ Tcell responses in hCD40Tg mice than in WT animals (not shown). Takentogether, the inventors concluded that CD40HVac targets human CD40 invivo and can thus elicit E6/7-specific cellular responses. The inventorsalso found that CD40HVac plus poly IC (adjuvant) was more potent thanCD40HVac alone at eliciting E6/7-specific T cell responses in hCD40Tgmice (data not shown), although humanized anti-CD40 antibody used inCD40HVac has an agonistic property.

CD40HVac plus poly IC can mount therapeutic immunity in hCD40Tg animals.hCD40Tg mice (10 mice per group) were SC challenged with HPVE6/7-expressing TC-1 tumor cells (5×10⁴). The inventors confirmed thatanimals harbor palpable tumors on day 6 after TC-1 challenge. Animalswere then immunized SC, intramuscularly (IM), or intraperitoneally (IP)with 30 μg CD40HVac plus 50 μg poly IC on days 6, 12, and 24. A controlgroup was kept without immunization. FIG. 9a shows that all animalsreceiving CD40HVac plus poly IC survived while all control animals died.Injection of poly IC alone did not promote survival (data not shown). Ina separate experiment, we measured progression of TC-1 tumors byassessing tumor volume (FIG. 9b ). All control animals (10 mice)developed tumors and died within 40 days of TC-1 challenge. In contrast,CD40HVac plus poly IC treatment suppressed tumor progression. It is alsoof note that some of the treated animals developed large tumors (200-600mm³), and these tumors regressed over time during vaccination. Takentogether, the inventors concluded that CD40HVac elicits therapeuticimmunity in hCD40Tg mice. Furthermore, the data indicated that the routeof immunization is an important factor that could impact the overalltherapeutic efficacy of the CD40HVac regimen.

CD8+ CTL infiltration into tumors is critical for tumor regression.Human CD40 transgenic (hCD40Tg) mice were SC challenged with highnumbers of TC-1 tumor cells (2×10⁵ cells). Animals were then immunizedwith 30 μg CD40HVac plus 50 μg poly IC on days 6 and 12. Withoutvaccination, all animals died within 25 days after the tumor challenge.On day 60, the percentages of H2-Db (RAHYNIVTF) tetramer+CD8+ T cells intumors and blood were assessed (FIG. 10). The percentage oftetramer+CD8+ T cells in the tumor (left) inversely correlates withtumor volume. There was no such correlation between the percentage oftetramer+CD8+ T cells in the blood (right) or spleen (not shown) and thetumor volume. Thus, infiltration of antigen-specific CD8+ CTLs intotumors is critical for tumor regression. Thus, we anticipate theimprovement of CD40HVac efficacy by promoting effector cell infiltrationinto and retention within mucosal tumors.

CD40HVac made with anti-CD40 (12E12 clone) is more efficient thanCD40HVac made with anti-CD40 (12B4 clone) at elicitingHPV16.E6/7-specific CD8+ T cell responses. The inventors comparedrecombinant fusion proteins made with three different clones ofanti-CD40 mAbs (12E12 and 12B4) for their ability to primeHPV16.E6/7-specific CD8+ T cell responses. The inventors used poly IC asan adjuvant. hCD40Tg animals received three doses of recombinant fusionproteins (30 μg/dose) plus poly IC (50 μg/dose) via s.c. Seven daysafter the third immunization, the percentage of E7-specific CD8+ T cellsin the blood were determined by tetramer staining. As shown in leftpanel of FIG. 11a , recombinant fusion proteins made with 12E12 was moreefficient than those made with 12B4 clone at inducing E6/7-specific CD8+T cell responses. The inventors also found that anti-CD40(12E12)-HPV16.E6/7 was more efficient than anti-CD40 (12B6)-HPV16.E6/7at eliciting IFNγ+CD8+ T cell responses by ELISPOT assay usingsplenocytes (left panel in FIG. 11b ). HPV16.E6/7 fused with the twoclones of anti-CD40 mAbs resulted in similar levels of E6/7-specificIFNγ+CD4+ T cell responses (right panel in FIG. 11b ).

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988-   Atherton et al., 1985-   Ausubel et al., 1996-   Barany and Merrifield, 1979-   Bird et al., 1988-   Burke et al., 1994-   Cumber et al., 1992-   Dholakia et al., 1989-   Epitope Mapping Protocols (1996)-   Glennie et al., 1987-   Goding, 1986, pp. 60 61-   Holliger et al, 1993-   Holliger & Winter, 1999-   Holt et al., 2003-   Hu et al. 1996-   Huston et al., 1988-   Khatoon et al., 1989-   King et al., 1989-   Kohl et al., 2003-   Kyte and Doolittle, 1982-   Liu et al., 2003-   McCafferty et al., 1990-   Merchand et al., 1998-   Merrifield, 1986-   O'Shannessy et al., 1987-   Owens & Haley, 1987-   Pack, et al., 1992-   Potter & Haley, 1983-   Reiter et al., 1996-   Repp et al., 1995-   Ridgeway et al., 1996-   Sambrook et al., 2001-   Skerra, 2000-   Skerra, 2001-   Staerz & Bevan, 1986-   Stewart and Young, 1984-   Tam et al., 1983-   Tigges et al., 1996-   Ward, 1989-   U.S. Pat. No. 3,817,837-   U.S. Pat. No. 3,850,752-   U.S. Pat. No. 3,939,350-   U.S. Pat. No. 3,996,345-   U.S. Pat. No. 4,196,265-   U.S. Pat. No. 4,277,437-   U.S. Pat. No. 4,275,149-   U.S. Pat. No. 4,366,241-   U.S. Pat. No. 4,472,509-   U.S. Pat. No. 4,554,101-   U.S. Pat. No. 4,879,236-   U.S. Pat. No. 4,938,948-   U.S. Pat. No. 5,196,066-   U.S. Pat. No. 5,741,957-   U.S. Pat. No. 5,750,172-   U.S. Pat. No. 5,756,687-   U.S. Pat. No. 5,827,690-   U.S. Pat. No. 5,871,986-   U.S. Pat. No. 6,091,001-   U.S. Pat. No. 6,651,655-   U.S. Patent Publication No. 2005/0106660-   U.S. Patent Publication No. 2006/0058510-   U.S. Patent Publication No. 2006/0088908-   U.S. Patent Publication No. 2010/0285564-   U.S. Patent Publication No. 2012/0039,916-   U.S. Patent Publication No. 2012/023,102-   U.S. Provisional Patent No. 61/332,465-   U.S. patent application Ser. No. 12/024,036-   U.S. patent application Ser. No. 12/024,897-   U.S. patent application Ser. No. 12/025,010-   U.S. patent application Ser. No. 12/026,095-   U.S. patent application Ser. No. 12/036,138-   U.S. patent application Ser. No. 12/036,158-   U.S. patent application Ser. No. 12/504,463-   U.S. patent application Ser. No. 12/717,778-   U.S. patent application Ser. No. 12/717,789-   U.S. patent application Ser. No. 12/717,804-   U.S. patent application Ser. No. 12/718,365-   U.S. patent application Ser. No. 12/882,052-   U.S. patent application Ser. No. 12/882,052-   U.S. patent application Ser. No. 13/100,684-   U.S. patent application Ser. No. 13/208,993-   U.S. patent application Ser. No. 13/269,951-   U.S. patent application Ser. No. 13/282,112-   U.S. patent application Ser. No. 13/415,564-   U.S. patent application Ser. No. 13/424,582-   U.S. patent application Ser. No. 13/430,206-   U.S. patent application Ser. No. 13/594,397-   U.S. patent application Ser. No. 13/596,526-   U.S. patent application Ser. No. 13/465,371-   U.S. patent application Ser. No. 13/397,932-   PCT Publication No. WO2006/056464-   PCT Publication No. WO94/13804-   PCT Publication No. WO 2008/103947-   PCT Publication No. WO 2008/103953-   PCT Publication No. WO 2008/118587-   PCT Publication No. WO 2010/104747-   PCT Publication No. WO 2010/104749-   PCT Publication No. WO 2010/104761-   PCT Publication No. WO 2012/021834-   PCT Patent Application No. PCT/US92/09965-   PCT Patent Application No. PCT/US13/72217-   PCT Patent Application No. PCT/US2013/05839

What is claimed is:
 1. One or more nucleic acids encoding a fusion protein comprising an anti-CD40 antibody or fragment thereof, comprising at least three complementarity determining regions from each of a heavy and light chain variable region of an anti-CD40 antibody, at least one peptide linker and at least one human papillomavirus (HPV) E6 or E7 antigen, wherein the E6 or E7 antigen or antigens are HPV type 16 or HPV type 18 antigens, wherein the at least one HPV E6 or E7 antigen comprises a peptide comprising the amino acid sequence of at least one of SEQ ID Nos: 1, 3 and
 4. 2. The one or more nucleic acids of claim 1, wherein the anti-CD40 antibody or fragment thereof is humanized.
 3. The one or more nucleic acids of claim 1, wherein the peptide linker or linkers comprise one or more glycosylation sites.
 4. The one or more nucleic acids of claim 1, wherein the peptide linker comprises one or both of Flexv1 (SEQ ID NO:5) and f1 (SEQ ID NO:6).
 5. The one or more nucleic acids of claim 1, wherein the fusion protein comprises one HPV E6 antigen of SEQ ID NO: 1 or 3 and one HPV E7 antigen of SEQ ID NO:2 or
 4. 6. The one or more nucleic acids of claim 1, wherein at least one nucleic acid comprises the sequence of SEQ ID NO:18.
 7. The one or more nucleic acids of claim 1, wherein at least one nucleic acid comprises the sequence of SEQ ID NO:20 or
 22. 8. The one or more nucleic acids of claim 1, wherein at least one HPV E6 antigen is an HPV type 16 antigen and at least one HPV E7 antigen is an HPV type 16 antigen.
 9. The one or more nucleic acids of claim 1, wherein at least one HPV E6 antigen is an HPV type 18 antigen and at least one HPV E7 antigen is an HPV type 18 antigen.
 10. The one or more nucleic acids of claim 1, wherein at least one HPV E6 antigen is an HPV type 16 antigen and at least one HPV E7 antigen is an HPV type 18 antigen or wherein and at least one HPV E6 antigen is an HPV type 18 antigen and at least one HPV E7 antigen is an HPV type 16 antigen.
 11. The one or more nucleic acids of claim 1, wherein the fusion protein comprises the amino acid sequences of at least SEQ ID NOs:11-13 and/or SEQ ID NOs:14-16.
 12. The one or more nucleic acids of claim 1, wherein the fusion protein comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 of SEQ ID Nos: 14-16, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 SEQ ID Nos:11-13, respectively.
 13. A vector comprising the one or more nucleic acids of claim
 1. 14. An isolated host cell comprising the one or more nucleic acids of claim
 1. 15. An isolated host cell comprising a nucleic acid encoding an anti-CD40 heavy chain variable region comprising at least three complementarity determining regions, at least one peptide linker and at least one human papillomavirus (HPV) type 16 or type 18 E6 or E7 antigen, wherein the at least one HPV E6 or E7 antigen comprises a peptide comprising the amino acid sequence of at least one of SEQ ID Nos: 1, 3 and
 4. 16. The cell of claim 15, wherein the cell further comprises a nucleic acid encoding an anti-CD40 light chain variable region comprising at least three complementarity determining regions.
 17. An isolated host cell comprising: i.) a nucleic acid comprising a sequence of SEQ ID NO:20, a nucleic acid that encodes a polypeptide of SEQ ID NO: 19, a nucleic acid comprising a sequence with at least 80% identity to SEQ ID NO:20, a nucleic acid that encodes a polypeptide with at least 80% identity to SEQ ID NO: 19, a nucleic acid comprising a sequence of SEQ ID NO:22, a nucleic acid that encodes a polypeptide of SEQ ID NO:21, a nucleic acid comprising a sequence with at least 80% identity to SEQ ID NO:22, or a nucleic acid that encodes a polypeptide with at least 80% identity to SEQ ID NO:21; and ii.) a nucleic acid comprising a sequence of SEQ ID NO: 18, a nucleic acid that encodes a polypeptide of SEQ ID NO: 17, a nucleic acid comprising a sequence with at least 80% identity to SEQ ID NO: 18, or a nucleic acid that encodes a polypeptide with at least 80% identity to SEQ ID NO:
 17. 18. A method of making a fusion protein comprising expressing the one or more nucleic acids of claim 1 in a host cell and isolating the fusion protein.
 19. A method of making a fusion protein comprising incubating the host cell of claim 16 and isolating the fusion protein. 